Diabetes-mediating proteins and therapeutic uses thereof

ABSTRACT

Provided are protective and deleterious diabetes-mediating proteins and polynucleotides encoding same, transgenic animals expressing a diabetes-mediating protein, drug screening methods for identifying a test compound capable of altering the expression of a diabetes-mediating protein, and methods of preventing or ameliorating diabetes by administering a compound capable of altering the expression of a diabetes-mediating protein.

This application is a 371 of PCT/IB9701627 filed Oct. 24, 1997, whichclaims the benefit under 35 USC 119(e) of earlier filed provisionalapplications: 60029324 filed 10/25/19996, 60030186 filed Nov. 5, 1996,and 60030088 filed Nov. 5, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to diabetes-mediating proteins, methodsof identifying diabetes-mediating proteins, transgenic animals useful inthe assays of the invention, methods for screening for drugs whichaffect the expression of diabetes-mediating proteins, and therapeuticcompounds for the treatment and prevention of diabetes.

2. Related Art

The development of insulin-dependent diabetes mellitus (IDDM) in man,and in animal models of human disease, is characterized by mononuclearcell infiltration and, β-cell destruction in the pancreatic islets(insulitis). The mechanisms behind β-cell destruction is not known.Accumulating evidence indicates that the cytokine interleukin-1,6(IL-1β), primarily produced by macrophages and monocytes, may be amediator of islet, β-cell destruction.

Animal models of human diabetes include diabetes-prone BB (BB-DP) ratsand non-obese diabetic (NOD) mice. 2-Dimensional (2D) gel maps of ratislet proteins have been constructed and used to determine qualitativeand quantitative changes in protein synthesis resulting with in vitroexposure of rat islet cells to IL-1β (Andersen et al. Diabetes44:400–407 (1995)).

Transgenic animal models of human diseases are known. For example, onemodel for human diabetes is a transgenic mouse expressing a viralprotein in the pancreatic β cells under control of the rat insulinpromoter (van Herrath et al. J. Clin. Invest. 98:1324–1331 (1996)). Lessthan 2% of the transgenic mice develop diabetes spontaneously; however,after a 2 month challenge with the virus, IDDM occurs in more than 95%of the mice.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery and identification ofdiabetes-mediating (DM) proteins. DM proteins are proteins which areinvolved in the development of diabetes or in the prevention of diabetesdevelopment in a subject at risk for the development of diabetes, andare identified by differential expression during the presence or absenceof disease development. The development of diabetes includes all stageswhich precede the clinically detectable stage.

Accordingly, in one aspect the invention features substantially purifieddiabetes-mediating proteins exhibiting an altered expression duringdevelopment of diabetes relative to expression in the absence ofdiabetes development. The purified diabetes-mediating proteins of theinvention are selected from the proteins listed in Tables 1 and 2. Noveldiabetes-mediating proteins are provided characterized by molecularweight, pI, and the mass spectroscopic characteristics as shown in FIGS.6–40. These proteins, referred to by their position on 10% IEF(isoelectric focusing) or NEPHGE (non-equilibrium pH-gradientelectrophoresis) 2-dimensional gels (FIGS. 1A–1B), are selected from thegroup consisting of NEPHGE 7, 9, 102, 123, 129, 130, 174, 181, 182, 211,231, 236, 253, 298, and IEF 665, 939,941,950, 1196.

DM proteins are further characterized as protective or deleterious DMproteins. A protective diabetes-mediating protein (“protective protein”)is characterized as a protein capable of protecting against thedevelopment of diabetes and/or delaying the onset of diabetes in asubject at risk for development of diabetes, or ameliorating thesymptoms of diabetes in a subject suffering from diabetes. A protectiveprotein may also be a protein which does not alter expression duringdevelopment of diabetes, but exhibits an altered expression in a subjectat risk for diabetes who escapes the development of diabetes. Adeleterious diabetes-mediating protein (“deleterious protein”) ischaracterized as a protein capable of enhancing the development ofdiabetes, increasing the risk of a subject developing diabetes, orreducing the time required for development of diabetes in a subject atrisk for development of diabetes. A deleterious protein may also be aprotein which does not alter expression during development of diabetes,but exhibits an altered expression in a subject at risk for diabetes whoescapes the development of diabetes. The diabetes-mediating protein ofthe invention may be identified by any means known to the art, includinggel electrophoresis, immunoblotting, mass spectrometry, orchromatography, and is characterized by an altered protein expressionduring development of diabetes as compared to the same protein expressedin the absence of diabetes development. U.S. provisional patentapplication Ser. No. 60/029,324 identifies proteins expressed inpancreatic islet cells identified by molecular weight and pI (FIGS. 1Aand 1B). The instant application provides a selection of these proteinswhich have been identified as diabetes-mediating proteins, as listed inTables 1 and 2, and FIGS. 6–40.

The diabetes-mediating proteins of the invention are useful in drugscreening assays for identifying compounds capable of modulating thedevelopment of diabetes, useful as therapeutic agents for the treatmentor prevention of diabetes, and useful as targets of therapeutic agentscapable of preventing or ameliorating diabetes by modulating theexpression of the diabetesmediating protein.

Changes in the expression of specific DM proteins is diagnosticallyuseful as indicative of the development of diabetes. Accordingly, in oneaspect the invention features a method for diagnosing the development ofdiabetes by measuring an increase in protein expression in one or moreproteins selected from the group consisting of the diabetes-mediatingproteins listed in Table 1, and a decrease in the protein expression ofone or more proteins selected from the list consisting of thediabetes-mediating proteins listed in Table 2. Changes in proteinexpression are measured in a test subject suspected of developingdiabetes or at risk for the development of diabetes and are expressedrelative to protein expression in a normal non-diabetes control. In apreferred embodiment, changes of combinations of one or more of theproteins of Tables 1 and 2 is indicative of the development of diabetes.In a more preferred embodiment, changes of a combination of 5 or more ofthe proteins of Tables 1 and/or 2 is indicative of the development ofdiabetes. In an even more preferred embodiment, changes of a combinationof 10 or more of the proteins of Tables 1 and/or 2 is indicative of thedevelopment of diabetes.

In one aspect, the invention features an in vivo assay method foridentifying proteins which are involved in the development of disease,e.g., diabetes. In a specific embodiment of the in vivo assay method ofthe invention, cells which secret insulin or are capable of developinginto insulin producing cells are transplanted to an immunologicallycompatible host animal which is an animal at risk for the development ofdiabetes. Protein expression is analyzed in transplanted cells rescuedat time points between the time of transplantation and disease onset,and proteins exhibiting an altered expression during disease developmentrelative to their expression in the absence of the development ofdiabetes are identified. In specific embodiments, the transplanted cellsare neonatal islet cells which are transplanted into a animal model atrisk for development of diabetes. In one specific embodiment, theneonatal islet cells are taken from neonatal BB-DP rats and the hostanimal is BB-DP rat. In another embodiment, the source of transplantedcells and host animals are NOD mice.

The invention provides identified diabetes-mediating proteins which maybe further characterized as protective or deleterious proteins. In oneembodiment of the invention, a candidate protective or deleteriousprotein is identified in vitro by transfecting cultured cells with apolynucleotide encoding the candidate protective or deleterious protein,and the effect of expression of the diabetes-mediating protein on invitro cell functionality upon challenge with IL-1β determined. Thepolynucleotide may be operably connected to an inducible promoter suchthat expression of the candidate protective or deleterious protein isunder exogenous control. Exogenous control may be exerted by agents,e.g., interferon, such agents being determined by the promoter selected.In specific embodiments, cell functionality is determined by measurementof nitric oxide (NO) production, insulin secretion, cell survival,and/or cytotoxicity upon exposure to IL-1β.

In an in vivo method for identifying protective or deleteriousdiabetes-mediating proteins, a transgenic mammal is generated expressingthe candidate protein, wherein the transgenic mammal is at risk fordeveloping diabetes, and the effect of transgene expression on thedevelopment and timing of diabetes onset is determined. A protectiveprotein is one which prevents, inhibits, or slows the development ofdiabetes in a subject at risk for diabetes, and a deleterious protein isone that causes the development of diabetes, increases the risk ofdevelopment of diabetes, or decreases the time required for thedevelopment of diabetes in a subject at risk for developing diabetes. Adeleterious protein is also a protein that prevents or interferes withthe expression of a protective protein.

The invention includes a substantially purified protective ordeleterious diabetes-mediating protein, and polynucleotide sequencewhich encodes the diabetes-mediating protein of the invention. In onenon-limiting embodiment, the protective protein is galectin-3 (FIGS. 4and 5) (SEQ ID NOs:1–2). In another non-limiting embodiment, thedeleterious protein is mortalin (FIGS. 2–3)(SEQ ID NOs:3–4).

In one aspect, the invention features a transgenic mammal having anexogenous diabetesmediating protein gene or genes inserted into itsgenome. The transgenic mammal of the invention is useful in assaymethods for determining the effect of the expression of adiabetes-mediating protein in the development of diabetes, and foridentifying protective or deleterious proteins. The transgene may be anatural, or partially or wholly artificial diabetes-mediating gene, andmay be different from or the same as an endogenous diabetes-mediatingprotein gene. In one embodiment, the transgene is under control of aninducible promoter.

In a related aspect, the invention features a transgenic mammal havingan exogenous deleterious gene, and exhibiting an increased incidence ofthe spontaneous development of diabetes within a predictable period oftime. In preferred embodiments, the transgenic mammal exhibits a greaterthan 50% chance, more preferably a greater than 60% chance, even morepreferably a greater than 70% chance, even more preferably a greaterthan 80% chance, and most preferably a greater than 90% chance ofdeveloping diabetes. In an embodiment of the invention, the transgenicmammal of the invention is transgenic for one or more genes encoding adeleterious diabetes-mediating protein. In another embodiment, thetransgenic mammal additionally has one or more endogenousdiabetes-mediating protein genes ablated. Generally, the transgenicmammal will have the transgenic gene under control of an insulin, CMV(cytomegalovirus), interferon, or MHC (myosin heavy chain) promoter. Infurther specific embodiments, a transgenic mammal expresses elevatedlevels of an endogenous diabetes-mediating gene obtained by an enhancedpromoter or a high copy number of an endogenous diabetes-mediatingprotein gene. In further specific embodiments, the transgenic mammal hasa disrupted diabetes-mediating protein gene.

The invention further includes mammals in which an endogenousdiabetes-mediating protein gene is exogenously altered by methods knownin the art, for example, by application of gene activation technologiessuch as that described in U.S. Pat. No. 5,641,670, entirely incorporatedherein by reference.

In one aspect, the invention features an assay for screening compoundswhich effect the expression of one or more diabetes-mediating proteins.In one embodiment, animals at risk for spontaneous development ofdiabetes are used in an assay for determining the ability of a testcompound to effect the expression of one or more diabetes-mediatingprotein(s). In a preferred embodiment, the assay animal is thetransgenic mammal of the invention having a high risk of the developmentof diabetes. By the term “effect the expression of a diabetes-mediatingprotein” is meant a compound which induces, enhances, inhibits, ordecreases the expression of an endogenous diabetes-mediating protein.

In specific embodiments, the invention provides an assay for identifyinga compound capable of inducing or enhancing the expression of anendogenous protective protein, and thus to delay or inhibit thedevelopment of diabetes. In another specific embodiment, the assaymethod of the invention is useful for identifying a compound capable ofsuppressing or inhibiting the expression of a deleteriousdiabetes-mediating protein, thus delaying or inhibiting the developmentof diabetes.

In a related aspect, the invention provides or an assay for identifyinga compound which modulates the activity of a diabetes-mediating protein,e.g., an agonist, an antagonist, or by blocking a post-translationalstep required for activation of a diabetes-mediating protein. Changes inthe expression of specific DM proteins are useful in a screening methodfor identifying compounds capable of modulating the expression of DMproteins. A compound which modulates the expression of one or morediabetes mediating proteins is useful as a potential therapeutic in thetreatment or prevention of diabetes. Accordingly, in one aspect theinvention features an assay method for identifying compounds capable ofmodulating the expression of diabetes-mediating proteins having thesteps of contacting a test compound with a cell or tissue expressing oneor more diabetes-mediating proteins, and determining the effect of thetest compound on the expression of one or more diabetes-mediatingproteins. Determination of the effect of a compound may be conducted bya variety of methods known to the art, including hybridization to probesor other oligonucleotides, antibody recognition, e.g., immunodiffusion,immunofluorescence, ELISA (Enzyme-Linked Immunosorbent Assay), RIA(radioimmunoassay), blotting, immunoprecipitation,immunoelectrophoresis, or chromatography, and electrophoresis. Acompound capable of increasing the expression of one or more proteinsselected from the group consisting of the diabetes-mediating proteinslisted in Tables 1 and 2 and decreasing the expression of one or moreproteins selected from the list consisting of the diabetes-mediatingproteins listed in Tables 1 and 2 is a candidate therapeutic agent forthe prevention or treatment of diabetes. Changes in protein expressionare determined relative to expression in the absence of the testcompound.

In another aspect, the invention provides a therapeutic method forpreventing diabetes in a subject at risk for diabetes or of amelioratingthe symptoms of diabetes in a diabetic subject by administering atherapeutically effective amount of a protective diabetes-mediatingprotein. Preferably the subject is a human. Also included in theinvention is gene therapy by providing a polynucleotide encoding aprotective diabetes-mediating protein. The invention further includes atherapeutic method for preventing and/or treating diabetes byadministering an effective amount of a polynucleotide which inhibits thein vivo expression of a deleterious diabetes-mediating protein.Candidate therapeutic compounds are selected from the proteins of Tables1 and 2.

In a related aspect, the invention provides a therapeutic method ofpreventing and/or treating diabetes in a subject at risk for diabetes byadministering a therapeutically effective amount of a compound capableof suppressing or reducing the expression of an endogenous deleteriousdiabetes-mediating protein. In another embodiment, the inventionprovides a therapeutic method of preventing and/or treating diabetes byadministering a therapeutically effective amount of a compound capableof inducing or enhancing the expression of an endogenous protectivediabetes-mediating protein. In a related aspect, the invention providesa therapeutic method of preventing and/or treating diabetes in a subjectat risk for diabetes by administering a therapeutically effective amountof a compound capable of modulating the activity of a diabetes-mediatingprotein, e.g., as an agonist, an antagonist, or by preventing theactivation of a diabetes-mediating protein. The therapeutic method ofthe invention includes ex vivo methods known to the art for providingthe therapeutic agent to a subject in need thereof.

An object of the invention is to identify proteins which mediatediabetes onset.

An object of the invention is to provide an in vivo assay foridentification of diabetesmediating proteins.

Another object of the invention is to provide diabetes-mediatingproteins which are useful in assays for identifying test compoundscapable of preventing, delaying, or ameliorating diabetes in a subject.

Another object of the invention is to provide transgenic animals usefulin assays to identify protective or deleterious diabetes-mediatingproteins.

Another object of the invention is to provide transgenic animals usefulin assay to identify test compounds capable of affecting the expressionof a diabetes-mediated protein.

Another object of the invention is to provide a transgenic host mammal(which is small, e.g., less than 1 kg when full grown, and inexpensiveto maintain) such as a mouse, rat or hamster which includes a natural,or partially or wholly artificial diabetes-mediating gene.

An advantage of the present invention is that the transgenic mammal canbe used to identify a protective protein which can prevent or inhibitdisease development in a manner which is substantially faster, moreefficient and cheaper than presently available assay methods.

Another advantage is that transgenic mammal can be used as test animalsfor testing drugs for efficacy in the treatment of humans suffering fromdiabetes or at risk for the development of diabetes.

Another object of the invention is to provide an assay foridentification of test compounds which effect the expression of adiabetes-mediating protein and which are capable of preventing the onsetof diabetes in a subject at risk for development of the disease, or forameliorating the symptoms of diabetes in a diabetic subject.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the diabetes-mediating gene(s) and protein(s), assay method,and transgenic mouse as more fully described below, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show a fluorograph of a 2-dimensional gel of proteinsexpressed in neonatal rat islet cells incubated for 24 h in RPMI1640+0.5% normal human serum, followed by a 4 h labeling with[³⁵S]-methionine. FIG. 1A is the isoelectric focusing gel (IEF; pH3.5–7). FIG. 1B is the non-equilibrium pH-gradient electrophoresis gel(NEPHGE; pH 6.5–10.5). Arrows mark 105 diabetes-mediating proteins.

FIG. 2 is the amino acid sequence of murine mortalin.

FIG. 3 is the amino acid sequence of human mortalin.

FIG. 4 is the amino acid sequence of rat galectin.

FIG. 5 is the amino acid sequence of human galectin-3.

FIG. 6 is the mass spectroscopy spectrum for diabetes-mediating proteinGR75 (mortalin), IEF Spot No. 340, determined using the parametersindicated on the figure legend.

FIG. 7 is the mass spectroscopy spectrum for diabetes-mediating protein,tentatively identified as lamin a, IEF Spot No. 655, determined asindicated.

FIGS. 8–10 are the mass spectroscopy spectrum for a diabetes-mediating,tentatively identified as pyruvate kinase, NEPHGE Spot No. 1, determinedas indicated.

FIG. 11 is the mass spectroscopy spectrum for a diabetes-mediating,tentatively identified as 6-phosphobructose-2-kinase, NEPHGE Spot No.169, determined as indicated.

FIG. 12 is the mass spectroscopy spectrum for a diabetes-mediating,tentatively identified as triose phosphate isomerase, NEPHGE Spot No.334, determined as indicated.

FIG. 13 is the mass spectroscopy spectrum for a diabetes-mediating,tentatively identified as fructose-biphosphase aldolase, NEPHGE Spot No.668, determined as indicated.

FIGS. 14–15 are the mass spectroscopy spectrum for a noveldiabetes-mediating protein, termed “IEF Spot No. 665,” having amolecular weight of 42,243 daltons and a pI of 5.82, determined asindicated.

FIG. 16 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed, “IEF Spot No. 939,” having a molecular weight of 25,851daltons and a pI of 5.09, determined as indicated.

FIGS. 17–18 are the mass spectroscopy spectrum for a noveldiabetes-mediating protein, termed “IEF Spot No. 941” having a molecularweight of 22,704 daltons and a pI of 5.15, determined as indicated.

FIG. 19 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “IEF Spot No. 950,” having a molecular weight of 25,753daltons and a pI of 4.53, determined as indicated.

FIGS. 20–23 are the mass spectroscopy spectrum for a noveldiabetes-mediating protein, termed “IEF Spot No. 1196” having amolecular weight of 143,064 daltons and a pI of 5.41, determined asindicated.

FIG. 24 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 7,” having a molecular weight of 65,522daltons and a pI of 7.28, determined as indicated.

FIG. 25 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 9,” having a molecular weight of115,709 daltons and a pI of 8.33, determined as indicated.

FIG. 26 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 102,” having a molecular weight of63,560 daltons and a pI of 7.26, determined as indicated.

FIG. 27 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 123,” having a molecular weight of57,040 daltons and a pI of 8.17, determined as indicated.

FIG. 28 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 129,” having a molecular weight of57,609 daltons and a pI of 7.72, determined as indicated.

FIG. 29 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 130,” having a molecular weight of55,734 daltons and a pI of 8.07, determined as indicated.

FIGS. 30–31 are the mass spectroscopy spectrum for a noveldiabetes-mediating protein, termed “NEPHGE Spot No. 174,” having amolecular weight of 53,830 daltons and a pI of 7.92, determined asindicated.

FIGS. 32–33 are the mass spectroscopy spectrum for a noveldiabetes-mediating protein, termed “NEPHGE Spot No. 181,” having amolecular weight of 49,422 daltons and a pI of 7.40, determined asindicated.

FIGS. 34–35 are the mass spectroscopy spectrum for a noveldiabetes-mediating protein, termed “NEPHGE Spot No. 182,” having amolecular weight of 54,098 daltons and a pI of 7.61, determined asindicated.

FIG. 36 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 211,” having a molecular weight of47,925 daltons and a pI of 7.28, determined as indicated.

FIG. 37 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 231,” having a molecular weight of44,362 daltons and a pI of 8.34, determined as indicated.

FIG. 38 is the mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 236,” having a molecular weight of43,162 daltons and a pI of 7.90, determined as indicated.

FIGS. 39–40 are the mass spectroscopy spectrum for a noveldiabetes-mediating protein, termed “NEPHGE Spot No. 253,” having amolecular weight of 39,106 daltons and a pI of 9.05, determined asindicated.

FIG. 41 is a mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “IEF Spot No. 831,” having a molecular weight of 34,600daltons and a pI of 4.76, determined as indicated.

FIGS. 42–43 are the mass spectroscopy spectrum for a noveldiabetes-mediating protein, termed “IEF Spot No. 949,” having amolecular weight of 26,800 daltons and a pI of 4.49, determined asindicated.

FIGS. 44–46 are the mass spectroscopy spectrum for a noveldiabetes-mediating protein, termed “NEPHGE Spot No. 129,” having amolecular weight of 57,600 daltons and a pI of 7.72, determined asindicated.

FIG. 47 is a mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 310,” having a molecular weight of35,800 daltons and a pI of 7.57, determined as indicated.

FIG. 48 is a mass spectroscopy spectrum for a novel diabetes-mediatingprotein, termed “NEPHGE Spot No. 326,” having a molecular weight of34,500 daltons and a pI of 8.62, determined as indicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present diabetes-mediating proteins and genes, assaymethodology, and transgenic used in the assay are described, it is to beunderstood that this invention is not limited to particular assaymethods, diabetes-mediating proteins and genes, test compounds, ortransgenic mammals described, as such methods, genes, preparations, andanimals may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to“diabetes-mediating protein” or “a diabetes-mediating protein” includemixtures of such diabetes-mediating proteins, reference to “theformulation” or “the method” includes one or more formulations, methods,and/or steps of the type described herein and/or which will becomeapparent to those persons skilled in the art upon reading thisdisclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

Definitions

The term “protein” includes proteins, polypeptides, and peptides whichare chains of amino acids, including all post-translationalmodifications (e.g., processing and truncations, glycosylations orphosphorylations) which often play decisive roles in modulating proteinfunction. The term also encompasses natural proteins as well assynthetic or recombinant proteins, polypeptides, and peptides.

The term “diabetes” includes insulin-dependent diabetes melitis (IDDM)and type I diabetes. The term “diabetes-related diseases” includes suchconditions as obesity, circulatory deficiencies, insulin-resistance,syndrome X, diabetic retinopathy, diabetic neuropathy, and theinvolvement of advanced glycation end products (AGE) in neuropathy andatherosclerosis.

The term “diabetes-mediating protein” means a protein which is involvedin the development of diabetes. A diabetes-mediating protein is aprotein which exhibits an altered expression during the development ofdiabetes, that is, a protein which is up- or down-regulated, or whoseexpression is modulated up or down, during the development of diabetes,as compared to the expression of the same protein in the absence of thedevelopment of diabetes. A diabetes-mediating protein also means aprotein that is modified as associated with the development of diabetesor diabetes-related diseases. Further, interleukin 1α (IL-1α) isexpected to have a detrimental effect on the insulin secreting islets ofLangerhans, both in vivo and in vitro and is considered to play a majorrole in the development of diabetes. Tthe treatment of islets causes themodulation of 106 proteins, up or down regulating their expression. Forthe purposes of this invention, the term “diabetes mediating protein” isdefined to also include all the proteins (and all of their modificationproducts) which have been demonstrated to be modulated by IL-α in ratislets in vitro, as further described in U.S. provisional applicationNos. 60/029,324 (filed Oct. 25, 1996), 60/030,186 (filed Nov. 5, 1996)and 60/030,088 (filed Nov. 5, 1996), the entire contents of which areincorporated herein by reference.

The term “protein modification” includes any change in structure (ie., aqualititive change) of a protein. Such modifications can include, butare not limited to, changes in the amino acid sequence, transcriptionalor translational splice variation, pre- or post-translationalmodifications to the DNA or RNA sequence, addition of macromolecules orsmall molecules to the DNA, RNA or protein, such as peptides, ions,vitamins, atoms, sugar-containing molecules, lipid-containing molecules,small molecules and the like, as well-known in the art.

One type of protein modification according to the present invention isby one or more changes in the amino acid sequence (substitution, deltionor insertion). Such changes could include, at one or more amino acids, achange from a charged amino acid to a different charged amino acid, anon-charged to a charged amino acid, a charged amino acid to anon-charged amino acid (e.g., giving rise to difference in pI orpossibly molecular weight). Any other change in amino acid sequence isalso included in the invention. The overall positional change in a gelof a modified protein with a changed amino acid sequence also depends onhow many overall charges there are in the protein, as known in the art.Changes in the resolution of the gel (e.g., changing the pH or othergradient component) of the gel can allow detection of minor or majoramino acid sequence changes. The type of analysis can also affect howchanges are detected, e.g., using sequencing, mass spectrometry, labeledantibody binding,

Another type of modification is by change in length, conformation ororientation in the protein-encoding DNA or RNA that affects the way theopen reading frame is read in the cell, which can give large changes inposition of the spot on the gel and which could affect the analysis ofthe protein type and position in the gel.

Another typd of protein modification is by changes in processing of theprotein in the cell. A non-limiting example is where some proteins havean “address label” specififying where in (or outside of) the cell theyshould be used. Such a label or tag can be in the form of a peptide, asugar or a lipid, which when added or removed from the protein,determines where the protein is located in the cell.

A further type of protein modification is due to the attachment of othermacromolecules to a protein. This group can include, but is not limitedto, any addition/removal of such a macromolecule. These molecules can beof many types and can be either permanent or temporary. Examplesinclude: (i) polyribosylation, (ii) DNA/RNA (single or double stranded);(iii) lipids and phosphlipids (e.g., for membrane attachment); (iv)saccharides/polysaccharides; and (v) glycosylation (addition of amultitude of different types of sugar and sialic acids in a variety ofsingle and branched structures so that the number of variations possibleis large).

Another type of protein modification is due to the attachment of othersmall molecules to proteins. Examples can include, but are not limitedto: (i) phosphorylation; (ii) acetylation; (iii) uridylation; (iv)adenylation; (v) methylation, and (vi) capping (diverse complexmodification of the N-terminus of the protein for assorted reasons).Most of these changes are often used to regulate a protein's activity.(v) and (vi) are also used to change the half-life of the proteinitself. These protein changes can be detected by 2D using severalmethods, such as labeling, changes in pI, antibodies or other specifictechniques directed to the molecules themselves, as known in the art.Molecular weight changes can be, but may not usually be detected by 2DGE(2-dimensional gel electrophoresis). MALD (matrix assisted laserdesorption of flight mass spectrometry) is preferred to detect andcharacterize these modifications.

The term “expression” is meant to include not only the physicalexpression of a protein, but also as a measure of the activity of anexpressed protein. For example, a protein can be expressed as aninactive form, which is activated by phosphorylation. While the actualexpression of the protein has not changed, its effective expression(activity) has been modified. On a gel, the change in activity may bemeasured as the change in expression of a modified form of the protein.

The term “affected protein” means a protein that is modified inexpression or modified structurally. An affected protein can thus be aprotein in which expression is modified due to treatment with one ormore compounds, a diseased or pathological state and/or an immunologicalchange in or outside the cell from which the protein is derived. Anaffected protein can alternatively or additionally also be a proteinwhich exhibits an altered expression as up- or down-regulated, or whoseexpression is modified in structure in any way that can be detected by amethod of the present invention, as compared to the the expression ofthe same protein (i.e., an “unaffected protein”) in the absence of suchtreatment, disease or immunological change.

The term “diabetes-mediating gene or polynucleotide” means geneticmaterial encoding a protein, peptide, or protein fragment which encodesan intact or fragment of a diabetes-mediating protein. The term includesany gene from any species which encodes a diabetes-mediating protein. Adiabetes-mediating gene or polynucleotide may be naturally occurring orpartially or wholly synthetic.

The term “substantially pure,” when referring to a polypeptide, means apolypeptide that is at least 60%, by weight, free from the proteins andnaturally-occurring organic molecules with which it is naturallyassociated. A substantially pure diabetes-mediating protein is at least75%, more preferably at least 90%, and most preferably at least 99%, byweight, diabetes-mediating protein. A substantially purediabetes-mediating protein can be obtained, by extraction from a naturalsource; by expression of a recombinant nucleic acid encoding adiabetes-mediating protein, or by chemically synthesizing the protein.Purity can be measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

As used herein, “polynucleotide” refers to a nucleic acid sequence ofdeoxyribonucleotides or ribonucleotides in the form of a separatefragment or a component of a larger construct. DNA encoding portions orall of the polypeptides of the invention can be assembled from cDNAfragments or from oligonucleotides that provide a synthetic gene whichcan be expressed in a recombinant transcriptional unit. Polynucleotidesequences of the invention include DNA, RNA, and cDNA sequences, and canbe derived from natural sources or synthetic sequences synthesized bymethods known to the art.

As used herein, an “isolated” polynucleotide is a polynucleotide that isnot immediately contiguous (i.e., covalently linked) with either of thecoding sequences with which it is immediately contiguous (i.e., one atthe 5′ end and one at the 3′ end) in the naturally-occurring genome ofthe organism from which the polynucleotide is derived. The termtherefore includes, for example, a recombinant polynucleotide which isincorporated into a vector, into an autonomously replicating plasmid orvirus, or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule independent of other sequences. It alsoincludes a recombinant DNA which is part of a hybrid gene encodingadditional polypeptide sequences.

The isolated and purified polynucleotide sequences of the invention alsoinclude polynucleotide sequences that hybridize under stringentconditions to the polynucleotide sequences specified herein. The term“stringent conditions” means hybridization conditions that guaranteespecificity between hybridizing polynucleotide sequences. One skilled inthe art can select posthybridization washing conditions, includingtemperature and salt concentrations, which reduce the number ofnonspecific hybridizations such that only highly complementary sequencesare identified (Sambrook et al. in Molecular Cloning, 2d ea.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), herebyspecifically incorporated by reference). For instance, such conditionsare hybridization under specified conditions, e.g. involving presoakingin 5×SSC and prehybridizing for 1 h at about 40° C. in a solution of 20%formamide, 5× Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and50 μg of denatured sonicated calf thymus DNA, followed by hybridizationin the same solution supplemented with 100 μM ATP for 18 h at about 40°C. (Sambrook et al. supra (1989)). The isolated and purifiedpolynucleotide sequences of the invention also include sequencescomplementary to the polynucleotide encoding a diabetesmediating protein(antisense sequences) and ribozymes.

The terms “host animal” and “host mammal” are used to describe animalsinto which donor cells are transplanted. Convenient host animals includemice, hamsters and rats.

The terms “ablated diabetes-mediating protein gene”, “disrupteddiabetes-mediating gene”, and the like are used interchangeably hereinto mean an endogenous diabetes-mediating protein gene which has beenaltered (e.g., add and/or remove nucleotides) in a manner so as torender the gene inoperative. It is also used to include ablation ormodification of controlling sequences or regulatory genes which alsorender the gene inoperative (partially or completely).

The term “increased risk of developing diabetes” and the like meananimals which are genetically predisposed to develop diabetes,preferably having a greater than 50% chance, more preferably a greaterthan 60% chance, even more preferably a greater than 70% chance, evenmore preferably a greater than 80% chance, and most preferably a greaterthan 90% chance to develop diabetes.

By “altered protein” or “altered protein expression” is meant proteinswhose expression is increased (“up regulated”), decreased (“downregulated”), inhibited (i.e., turned off), or induced (i.e., turned on)during the development of diabetes.

By the term “modulating the activity” or the like is meant altering theactivity of a protein to prevent or enhance its normal activity, e.g.,as an agonist, antagonist, or by blocking a post-translationalmodification step required for protein activity.

By the term “effective amount” or “therapeutically effective amount” ismeant an amount of a compound sufficient to obtain the desiredphysiological effect, e.g., suppression of or delay of the developmentof diabetes.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may by prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, particularly a human, andincludes:

(a) preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

(b) inhibiting the disease, i.e., arresting its development; or

(c) relieving the disease, i.e., causing regression of the disease. Theinvention is directed to treating patients with or at risk fordevelopment of diabetes and related conditions mediated diabetes,insulin insufficiency, or insulin resistance. More specifically,“treatment” is intended to mean providing a therapeutically detectableand beneficial effect on a patient at risk for or suffering fromdiabetes.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down thedevelopment of a disease. Those in need of treatment include thosealready with the disorder as well as those prone to have the disorder orthose in which the disorder is to be prevented.

The terms “synergistic”, “synergistic effect” and like are used hereinto describe improved treatment effects obtained by combining one or moretherapeutic agents. Although a synergistic effect in some field is meantan effect which is more than additive (e.g., 1+1=3), in the field ofmedical therapy an additive (1+1=2) or less than additive (e.g.,1+1=1.6) effect may be synergistic. For example, if each of two drugswere to inhibit the development of diabetes by 50% if givenindividually, it would not be expected that the two drugs would becombined to completely stop the development of diabetes. In manyinstances, due to unacceptable side effects, the two drugs cannot beadministered together. In other instances, the drugs counteract eachother and slow the development of diabetes by less than 50% whenadministered together. Thus, a synergistic effect is said to be obtainedif the two drugs slow the development of diabetes by more than 50% whilenot causing an unacceptable increase in adverse side effects.

Abbreviations used herein include: IDDM=insulin dependent diabetesmellitus; BB-DP=diabetes prone Bio-Breeding rats; NOD=non-obese diabeticmice.

GENERAL ASPECTS OF THE INVENTION

The present invention encompasses several aspects including: (1)diabetes-mediating proteins identified by differential expression in thepresence and absence of the development of diabetes; (2) patterns andcombinations of DM proteins useful for predicting the development ofdiabetes and for identifying a compound able to effect a combination ofDM proteins in a desired manner; (3) protective diabetes-mediatingproteins; (4) deleterious diabetes-mediating proteins; (5) a method todiagnose for the onset or development of diabetes based on the detectionof one or more of the DM proteins, their post-translational modificationor degradation products in a body fluid; (6) an in vivo method foridentifying a diabetes-mediating protein; (7) a transgenic mammalcontaining an exogenous gene encoding a diabetes-mediating protein; (8)a transgenic mammal useful in an in vivo assay for identifyingprotective or deleterious diabetes-mediating proteins; (9) an in vitroassay using transduced cultured cells expressing a diabetes-mediatingprotein useful for identifying protective or deleteriousdiabetes-mediating proteins; (10) an in vivo assay using transducedislet cells expressing a diabetes-mediating protein useful foridentifying protective or deleterious diabetes-mediating proteins; (11)an improved animal model for human diabetes, which animal exhibits ahigh incidence of diabetes within a predictable period of time; (12) invivo assay methods for identifying test compounds capable of inducing orenhancing the expression of one or more protective proteins; (13) invivo assay methods for identifying test compounds capable of inhibitingor reducing the expression of deleterious proteins, (14) methods fortreating diabetes and/or preventing or slowing the development ofdiabetes in a mammal by providing a therapeutically effective amount ofa protective diabetes-mediating protein; (15) methods for treatingdiabetes and/or preventing or slowing the development of diabetes in amammal by providing a therapeutically effective amount of a compoundcapable of inducing or enhancing the expression of a protectivediabetes-mediating protein or of modulating the activity of adiabetesmediating protein; and (16) methods for treating diabetes and/orpreventing or slowing the development of diabetes in a mammal byproviding a therapeutically effective amount of a compound capable ofinhibiting or reducing the expression of a deleteriousdiabetes-mediating protein, or of modulating the activity of adiabetes-mediating protein.

I. Diabetes-Mediating Proteins, Polypeptides, Polynucleotides, andAntibodies.

The invention provides diabetes-mediating proteins, that is, proteinsidentified as involved in or effected during the development ofdiabetes. Diabetes-mediating proteins are characterized as proteinswhose expression is altered during the development of diabetes relativeto their expression in the absence of the development of diabetes. Thepresent disclosure identifies diabetes-mediating proteins from a2-dimensional gel database of pancreatic islet cell proteins.Diabetes-mediating proteins include protective diabetes-mediatingproteins and deleterious diabetes-mediating proteins.

The invention provides in vitro methods for identifyingdiabetes-mediating proteins, including by functionally assessment byexpression of cloned cDNA as sense or antisense constructs intransfected cells to establish their deleterious or protective role incytokine-mediated cytotoxicity. B cells of the islets of Langerhans arespecifically sensitive to the toxic effect of cytokines. It haspreviously been demonstrated that lipofection of rat β cells with heatshock protein 70 (HSP70) and induction of hemeoxygenase (HO) by exposureto hemin improved in vitro survival of cells exposed to IL-1 (Karlsen etal. in: Insulin Secretion and Pancreatic B Cell Research, ed: P. R.Flatt and S. Lenzen; Smith-Gordon, USA, pp. 499–507 (1994)). Cells maybe transfected in a number of ways known to the art, for example, theadenoviral vector method. See, for example, Korbutt et al.Transplantation Proceedings 27:3414 (1995); Csete et al. 26:756–757(1994); Becker et al. J. Biol. Chem. 269:21234–21238 (1994).

Following establishment of stable transfected β cell clones, the effectof the expression of a diabetes-mediating protein may be determined byfunctional analysis of the clones cultured in the absence and presenceof cytokines. The functional analyses include nitrous oxide production(NO) measured as nitrite (Green et al. Anal. Biochem. 126:131–138(1982)), insulin secretion (Id.), cytotoxicity, and 2D-gelelectrophoresis. Cytotoxicity may be measured by a variety of methodsknown to the art, including (1) a calorimetric assay based on lactatedehydrogenase (LDH) release (CytoTox, Promega), (2) a life-death assaybased on calcein uptake and fluorescence of living cells and ethidiumbromide staining of the nuclei of dead cells (Molecular Probes), (3)non-radioactive cell proliferation assay (MTT, Promega), apoptosis(Nerup et al. in: IDDM, S. Baba & T. Kaneko, eds., Elsevier Science, pp.15–21 (1994)), and/or semiquantitative multiplex PCR analysis of geneexpression. 2-dimensional gels can be used to compare control andcytokine stimulated islets to identify which proteins respond,identifying the proteins which play a role in the cell response.Interlink analysis can be used to define functional groups of proteinsand their regulation (e.g., by kinase phosphorylation or otherpost-translational modifications).

Preferred cells for use in the in such an assay are insulin-secretingcells, for example, MSL or RIN cells. The MSL cell line is a pluripotentor stem-cell like metastatic rat insulinoma cell line. Dependent uponculture and/or passage conditions in vitro and in vivo, the MSL cell canacquire all four hormone secretory phenotypes characteristic of theislets of Langerhans (Mandrup-Poulsen et al. Eur. J. Endocrinology133:660–671 (1995); Id. Eur. J. Endocrinology 134:21–30 (1996)). RINcells are a cultured line of insulinoma cells (Nielsen Exp. Clin.Endocrinol. 93:277–285 (1989)).

Protective diabetes-mediating proteins. The invention providessubstantially purified protective diabetes-mediating proteins(“protective proteins”) characterized as capable of protecting againstdevelopment of diabetes in a subject at risk for the development of thedisease or ameliorating or reducing the symptoms of diabetes in asubject suffering from diabetes. The protective protein of the inventionmay act directly to protect against diabetes, or may act indirectly byinducing or increasing the synthesis of a second protective protein orby reducing or inhibiting the synthesis of a deleterious protein.

In a specific embodiment, the invention provides the substantially pureprotective protein galectin-3. The sequence of rat galectin (SEQ IDNO:3) is shown in FIG. 4, and for human galectin-3(SEQ ID NO:4) in FIG.5. As shown below, gal-3 expressed in transfected cells increased cellsurvival upon challenge with IL-1β. Galectins are lectins withspecificity for β-galactoside sugars or glycoconjugates which arepresent in fetal and adult pancreatic islet cells. The term“substantially pure” as used herein refers to gal-3 which issubstantially free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated. One skilled in the artcan purify gal-3 using standard techniques for protein purification. Thepurity of the gal-3 polypeptide can also be determined by amino-terminalamino acid sequence analysis. The gal-3 protein includes functionalfragments of the polypeptide, as long as the protective activityremains. Smaller peptides containing the biological activity of gal-3are included in the invention. The invention further includes amino acidsequences having at least 80%, preferably 90%, more preferably 95%identity to the fully length amino acid sequence of SEQ ID NO:4. Percenthomology or identity can be determined, for example, by comparingsequence information using the GAP computer program, version 6.0,available from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch J. Mol. Biol. 48:443 (1970), as revised by Smith and WatermanAdv. Appl. Math. 2:482 (1981). Briefly, the GAP program definessimilarity as the number of aligned symbols (i.e., nucleotides or aminoacids) which are similar, divided by the total number of symbols in theshorter of the two sequences. The preferred default parameters for theGAP program include: (1) a unitary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) and the weightedcomparison matrix of Gribskov and Burgess Nucl. Acids Res. 14:6745(1986), as described by Schwartz and Dayhoff, eds. Atlas of proteinSequence and Structure, National Biomedical Research Foundation, pp.353–358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10penalty for each symbol in each gap; and (3) no penalty for end gaps.

The invention also provides purified proteins identified orcharacterized by a computer system or method of the present invention,where the protein can be selected from the group consisting of

-   -   (a) unaffected proteins having the corresponding molecular        weights and pIs as presented in Table 8;    -   (b) affected proteins having the corresponding molecular weights        and pIs as presented in Table 9; and    -   (c) marker proteins having the corresponding molecular weights        and pIs as presented in Table 10,    -   and wherein said at least one of said proteins is optionally        further selected from the group consisting of    -   (i) unaffected proteins having the corresponding molecular        weights and pIs as presented in Table 11;    -   (ii) affected proteins having the corresponding molecular        weights and pIs as presented in Table 12; and    -   (iii) marker proteins having the corresponding molecular weights        and pIs as presented in Table 13.

According to the invention, an affected or unaffected peptide includesan association of two or more polypeptide domains, such astransmembrane, cytoplasmic, hydrophobic, hydrophilic, ligand binding, orpore lining domains, or fragments thereof, corresponding to an affectedor unaffected peptide, such as 1–40 domains or any range or valuetherein. Such domains of an affected or unaffected peptide of theinvention can have at least 74% homology, such as 74–100% overallhomology or identity, or any range or value therein to one or morecorresponding affected or unaffected protein or peptide domains asdescribed herein. As would be understood by one of ordinary skill in theart, the above configuration of domains are provided as part of anaffected or unaffected peptide of the invention, such that a functionalaffected or unaffected protein or peptide, when expressed in a suitablecell, is capable of the associated biological activity found in thataffected islet cell type. Such activity, as measured by suitableaffected or unaffected protein or peptide activity assays, establishesaffected or unaffected protein or peptide activity of one or moreaffected or unaffected proteins or peptides of the invention.

Accordingly, an affected or unaffected peptide of the inventionalternatively includes peptides having a portion of an affected orunaffected protein or peptide amino acid sequence which substantiallycorresponds to at least one 20 to 10,000 amino acid fragment and/orconsensus sequence of an affected or unaffected peptide, or group ofaffected or unaffected peptides, wherein the affected or unaffectedprotein or peptide has homology or identity of at least 74–99%, such as88–99% (or any range or value therein, e.g., 87–99, 88–99, 89–99, 90–99,91–99, 92–99, 93–99, 94–99, 95–99, 96–99, 97–99, or 98–99%) homology oridentity to at least one sequence or consensus sequence of at least oneprotein characterized as presented in one or more of tables 8–13, or aspresented in FIGS. 2–7, having the mass spec characteristics of one ormore of proteins according to the present invention.

In one aspect, such an affected or unaffected peptide can maintainaffected or unaffected protein or peptide biological activity. It ispreferred that an affected or unaffected peptide of the invention is notnaturally occurring or is naturally occurring but is in a purified orisolated form which does not occur in nature. Preferably, an affected orunaffected peptide of the invention substantially corresponds to any setof domains of an affected or unaffected protein or peptide of theinvention, having at least 10 contiguous amino acids of proteinscharacterized in one or more of tables 8–13, comprising SEQ ID NOS:,having the mass spec characteristics of one or more of proteinsaccording to the present invention.

Alternatively or additionally, an affected or unaffected peptide of theinvention can comprise at least one domain corresponding to knownprotein domains, such as cytoplasmic, intracellular, transmembrane,extracellular, or other known domains, having 74–100% overall homologyor any range or value therein. Alternative domains are also encoded byDNA which hybridizes under stringent conditions to at least 30contiguous nucleotides encoding at least 10 contiguous amino acids ofproteins characterized in one or more of tables 8–13, FIGS. 2–7,comprising SEQ ID NOS:, having the mass spec characteristics of one ormore of of proteins according to the present invention, or at least 74%homology thereto, or having codons substituted therefor which encode thesame amino acid as a particular codon. Additionally, phosphorylation(e.g., PKA and PKC) domains, as would be recognized by the those skilledin the art are also considered when providing an affected or unaffectedpeptide or encoding nucleic acid according to the invention. Anon-limiting example of this is presented in proteins 672–674 of table12, wherein the same protein is differentially phosphorylated.

The invention further includes polynucleotide sequences encoding thediabetes-mediating proteins of the invention, including DNA, cDNA, andRNA sequences. It is also understood that all polynucleotides encodingall or a portion of a diabetes-mediating protein are also includedherein, as long as they encode a polypeptide with the diabetes-mediatingactivity. Such polynucleotides include naturally occurring, synthetic,and intentionally manipulated polynucleotides. For example, such apolynucleotide may be subjected to site-directed mutagenesis. Thepolynucleotide sequences of the invention also include antisensesequences. Antisense sequences include sequences synthesized withmodified oligonucleotides. The polynucleotides of the invention includesequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than one**codon. Therefore, all degenerate nucleotide sequences are included inthe invention as long as the amino acid sequence of thediabetes-mediating polypeptide is encoded by the nucleotide sequence isfunctionally unchanged.

The DNA sequences of the invention can be obtained by several methods.For example, the DNA can be isolated using hybridization techniqueswhich are well known in the art. These include, but are not limitedto: 1) hybridization of genomic or cDNA libraries with probes to detecthomologous nucleotide sequences, 2) polymerase chain reaction (PCR) ongenomic DNA or cDNA using primers capable of annealing to the DNAsequence of interest, and 3) antibody screening of expression librariesto detect cloned DNA fragments with shared structural features.

Preferably the DNA sequences of the invention is derived from amammalian organism, and most preferably from a human. Screeningprocedures which rely on nucleic acid hybridization make it possible toisolate any gene sequence from any organism, provided the appropriateprobe is available. Oligonucleotide probes, which correspond to a partof the sequence encoding the protein in question, can be synthesizedchemically. This requires that short, oligopeptide stretches of aminoacid sequences must be known. The DNA sequence encoding the protein canbe deduced from the genetic code, however, the degeneracy of the codemust be taken into account. The codon bias of the organism can be takeninto account to select the most probable nucleotide triplets. It ispossible to perform a mixed addition reaction when the sequence isdegenerate. This includes a heterogeneous mixture of denatureddouble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Sambrook et al. Molecular Cloning: ALaboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press,Plainview, N.Y. (1989)).

The development of specific DNA sequences encoding thediabetes-mediating proteins of the invention can also be obtained by: 1)isolation of double-stranded DNA sequences from the genomic DNA; 2)chemical manufacture of a DNA sequence to provide the necessary codonsfor the polypeptide of interest; and 3) in vitro synthesis of adouble-stranded DNA sequence by reverse transcription of mRNA isolatedfrom a eukaryotic donor cell. In the latter case, a double stranded DNAcomplement of mRNA is eventually formed which is generally referred toas cDNA. Of the three above-noted methods for developing specific DNAsequences for use in recombinant procedures, the isolation of genomicDNA isolates is the least common. This is especially true when it isdesirable to obtain the microbial expression of mammalian polypeptidesdue to the presence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression for theprotein of interest. When used in combination with polymerase chainreaction technology, even rare expression products can be cloned. Inthose cases where significant portions of the amino acid sequence of thepolypeptide are known, the production of labeled single ordouble-stranded DNA or RNA probe sequences duplicating a sequenceputatively present in the target cDNA may be employed in DNA/DNAhybridization procedures which are carried out on cloned copies of thecDNA which have been denatured into a single-stranded form (Jay et al.Nucl. Acid Res., 11:2325 (1983)).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for diabetes-mediating peptides having at least one epitope,using antibodies specific for a diabetes-mediating protein. Suchantibodies can be either polyclonally or monoclonally derived and usedto detect expression product indicative of the presence of the desiredcDNA.

DNA sequences encoding a diabetes-mediating can be expressed in vitro byDNA transfer into a suitable host cell. “Host cells” are cells in whicha vector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart.

In the present invention, the polynucleotide sequences encodingdiabetes-mediating proteins may be inserted into a recombinantexpression vector. The term “recombinant expression vector” refers to aplasmid, virus or other vehicle known in the art that has beenmanipulated by insertion or incorporation of the X130 genetic sequences.Such expression vectors contain a promoter sequence which facilitatesthe efficient transcription of the inserted genetic sequence of thehost. The expression vector typically contains an origin of replication,a promoter, as well as specific genes which allow phenotypic selectionof the transformed cells. Vectors suitable for use in the presentinvention include, but are not limited to the T7-based expression vectorfor expression in bacteria (Rosenberg et al. Gene 56:125 (1987)), thepMSXND expression vector for expression in mammalian cells (Lee andNathans J. Biol. Chem. 263:3521 (1988)) and baculovirus-derived vectorsfor expression in insect cells. The DNA segment can be present in thevector operably linked to regulatory elements, for example, a promoter(e.g., T7, metallothionein I, or polyhedron promoters).

Polynucleotide sequences encoding a diabetes-mediating protein can beexpressed in either prokaryotes or eukaryotes. Hosts can includemicrobial, yeast, insect and mammalian organisms. Methods of expressingDNA sequences having eukaryotic or viral sequences in prokaryotes arewell known in the art. Biologically functional viral and plasmid DNAvectors capable of expression and replication in a host are known in theart. Such vectors are used to incorporate DNA sequences of theinvention.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the diabetes-mediating proteinof the invention, and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector, such as simian virus40 (SV40) or bovine papilloma virus, to infect, transform, or transduceeukaryotic cells and express the protein (see, for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ea., 1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.2DGE is a preferred method for purification of modifications variants ofthe proteins from each other.

Deleterious diabetes-mediating proteins. Deleterious diabetes-mediatingproteins (“deleterious proteins”) are characterized as enhancing thedevelopment of or increasing the risk of a subject developing diabetes.The invention includes substantially purified protectivediabetes-mediating proteins, and polynucleotide sequences encoding suchproteins. In a preferred embodiment, a deleterious protein is mortalin.The amino acid sequence of murine mortalin (SEQ ID NO:1) is shown inFIG. 2, and of human mortalin (SEQ ID NO:2) in FIG. 3.

Antibodies specific to diabetes-mediatina proteins. Thediabetes-mediating proteins of the invention can also be used to produceantibodies which are immunoreactive or bind to epitopes of the diabetesmediating proteins. An antibody may consist essentially of pooledmonoclonal antibodies with different epitopic specificities, as well asdistinct monoclonal antibody preparations. Monoclonal antibodies aremade from antigen containing fragments of the protein by methods wellknown in the art (Kohler et al. Nature 256:495 (1975); Current Protocolsin Molecular Biology, Ausubel et al., ea., 1989).

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)2, and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with its antigen or receptor andare defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)2, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)2 is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. See, forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference.

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibodies which bind to the diabetes-mediating polypeptides of theinvention can be prepared using an intact polypeptide or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or a peptide used to immunize an animal can be derived fromtranslated cDNA or chemical synthesis which can be conjugated to acarrier protein, if desired. Such commonly used carriers which arechemically coupled to the peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.The coupled peptide is then used to immunize the animal (e.g., a mouse,a rat, or-a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elusion from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies. See, for example, Coliganet al. Unit 9, Current Protocols in Immunology, Wiley Interscience(1994), herein specifically incorporated by reference.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

For purposes of the invention, an antibody or nucleic acid probespecific for a diabetes-mediating protein may be used to detect thediabetes-mediating protein (using antibody) or encoding polynucleotide(using nucleic acid probe) in biological fluids or tissues. The antibodyreactive with the diabetes-mediating protein or the nucleic acid probeis preferably labeled with a compound which allows detection of bindingto the diabetes-mediating protein. Any specimen containing a detectableamount of antigen or polynucleotide can be used. Further, specificproteins may be selected by their ligands, e.g., galectin-3 binding tolectin-binding protein), and is useful for diagnosis and/or purificationof the protein of interest.

When the cell component is nucleic acid, it may be necessary to amplifythe nucleic acid prior to binding with a diabetes-mediatingprotein-specific probe. Preferably, polymerase chain reaction (PCR) isused, however, other nucleic acid amplification procedures such asligase chain reaction (LCR), ligated activated transcription (LAT) andnucleic acid sequence-based amplification (NASBA) may be used.

II. Method for Identifying a Diabetes-Mediating Protein

In vivo animal transplantation model. The invention features a methodfor identifying a diabetes-mediating protein, by providing an in vivoassay system which detects proteins which are involved in thedevelopment of diabetes or which are specifically effected duringdevelopment of diabetes. The in vivo transplantation assay of theinvention described herein allows the identification of proteinsinvolved or effected.

In the method for identifying a diabetes-mediating protein of theinvention, insulin-secreting cells or cells capable of developing intoinsulin-secreting cells are transplanted into a host animal.Transplanted cells are rescued at time points between transplantationand the onset of diabetes, and protein expression determined, andprotein expression compared with nontransplanted islets and syngeneictransplants in animals not developing diabetes. The method of theinvention allows proteins exhibiting an altered expression duringdevelopment of diabetes to be identified. Identified proteins are thenisolated and tested further to identification as protective ordeleterious diabetes-mediating proteins.

Cells capable of developing into insulin-producing cells includepancreatic islet cells and β cells. Transplanted cells may be obtainedfrom any species of interest, including human cells. A host animal ispreferably one which is immunologically compatible with the transplantedcells such that the transplanted cells do not undergo rejection in thehost animal.

The host animal may be any animal which develops diabetes and isconvenient for study. Preferred host animals are mice, rats andhamsters, with rats and mice being most preferred. In particularlypreferred embodiments, the host animal is selected from a strain bredfor an increased incidence of diabetes, including BB-DP rats and NODmice. The method of the invention may also be used with host animalsengineered to develop diabetes at a predetermined time, e.g., uponexposure to a specific antigen. See, for example, Oldstone et al. APMIS104:689–97 (1996); von Herrath et al. J. Clin. Invest. 98:1324–1331(1996); Morgan et al. J. Immunol. 157:978983 (1996). Other possible hostanimals include those belonging to a genus selected from Mus (e.g.mice), Rattus (e.g. rats), Oryctolagus (e.g. rabbits), and Mesocricetus(e.g. hamsters) and Cavia (e.g., guinea pigs). In general mammals with anormal full grown adult body weight of less than 1 kg which are easy tobreed and maintain can be used.

Method of determining protein expression. Protein expression may beassessed by a variety of means known to the art, including one ortwo-dimensional gel electrophoresis and immunoblotting.

Two-dimensional gel electrophoresis (2-DGE) is a particularly effectivetool for separating mixtures of proteins (Andersen et al. Diabetes44:400–407 (1995)). Cell protein extracts are put onto a gel, and theindividual proteins are separated first by charge and then by size. Theresult is a characteristic picture of as many as 1,000 to 5,000 spots,each usually a single protein. Resolution is improved by increasing gelsize, and by enhancing the sensitivity trough the use of radiolabelmethods, silver staining, and the reduction in thickness of the gels to1.5 mm and less.

Method for isolating diabetes-mediating proteins. As described in theExamples below, single proteins recovered from 2D gels can be identifiedby mass spectrometry to obtain a trypsin cleavage pattern as well as theprecise molecular weight of each peptide. These observed values are thenused to search in DNA and protein databases to determine if matchesexist to previously identified proteins. Identity can be determined froma known protein or deduced from high homology to a known protein. When2D gel electrophoresis is used to separate and identify protein spotswhich exhibit an altered synthesis during development of diabetes, anidentified protein spot is excised from the gel and digested withtrypsin to produce peptides. The peptides are recovered from the gel andsubjected to mass spectroscopy (matrix assisted laserdesorption/ionization mass spectrometry)(MALDI) and the resultingMS-profiles are analyzed against the computerized MS-profiles of allsequences found in the public sequence databases, as well as againstpropriety sequence information. If any matches to previously clonedsequences are obtained, information about the corresponding gene andencoded protein is collected. When an identified diabetes-mediatingprotein does not match a previously cloned protein, the protein may bemicrosequenced to obtain partial amino acid sequence information bymethods known to the art (see Example 5 below). Based upon resultsobtained from database searches or amino acid sequencing, specific ordegenerate primers are constructed and used to screen rat and humanislets libraries or first-strand cDNA by PCR is used to clone partialsequences of the corresponding cDNA. The obtained sequences are thenused to obtain full-length coding regions either by 5′-race PCR or byconventional hybridization screening techniques, followed by expressionof the recombinant protein (Karlsen et al. Proc. Natl. Acad. Sci. USA88:8337–8341 (1991); Katisen et al. in: Insulin secretion and pancreaticB-cell research, Flatt, P. R., ea., Smith-Gordon, USA; Chapter 64, pp.1–9 (1994); Karlsen et al. Diabetes 44:757–758 (1995).

Diabetes-mediating proteins can be isolated in a variety of ways knownto the art, including purification from biological material, expressionfrom recombinant DNA (see above). Conventional method steps includeextraction, precipitation, chromatography, affinity chromatography, andelectrophoresis. For example, cells expressing a diabetes-mediatingprotein can be collected by centrifugation, or with suitable buffers,lysed, and the protein isolated by column chromatography, for example,on DEAE-cellulose (diethylaminoethylcellulose), phosphocellulose,polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis orimmunoprecipitation. Diabetes-mediating proteins may alternatively beisolated by immunoprecipitation with the use of specific antibodies.

III. Transgenic Animals Expressing a Diabetes-Media Ting ProteinTransgene.

In one aspect, the invention includes a transgenic animal containing agene encoding a diabetes-mediating protein, as well as transgenicanimals which are the offspring of a transgenic animal of the invention.The gene encoding a diabetes-mediating protein may be comprised of anaturally occurring or partially or completely of an artificialpolynucleotide sequence, i.e. codon sequences not present in the nativegene sequence. Transgenic animals containing elevated levels ofexpression of the diabetes-mediating gene of the invention can beobtained for example, by over-expression of the gene with an enhancedpromoter and/or with high copy numbers of the natural gene. Transgenicanimals also specifically include a hybrid transgenic animal produced bycrossing a transgenic animal with an animal in which one or morediabetes-mediating protein gene(s) are ablated.

Transgenic animals of the invention are useful in a number of ways,including in assays for determining the effect of a candidate protectiveor deleterious diabetes-mediating protein on the development ofdiabetes. For example, a transgenic animal carrying a transgene for acandidate protective protein is useful for determining the effect of theexpression of the protective transgene on the development of diabetes.

Preferred host animals are mice, rats and hamsters, with rats and micebeing most preferred in that there exists considerable knowledge on theproduction of transgenic animals. Other possible host animals includethose belonging to a genus selected from Mus (e.g. mice), Rattus (e.g.rats), Oryctolagus (e.g. rabbits), and Mesocricetus (e.g. hamsters) andCavia (e.g., guinea pigs). In general mammals with a normal full grownadult body weight of less than 1 kg which are easy to breed and maintaincan be used.

The transgenic non-human mammal of the invention (preferably a rat ormouse) will have in some or all of its nucleated cells a gene encoding adiabetes-mediating protein, e.g., one or more of a protection protein ordeleterious protein, which gene was introduced into the mammal, or anancestor of that mammal, at an embryonic or germ cell stage. This“embryonic stage” may be any point from the moment of conception (e.g.,as where the sperm or egg bears the foreign gene) throughout all of thestages of embryonic development of the fetus. A “transgenic mammal” asused herein denotes a mammal bearing in some or all of its nucleatedcells one or more genes derived from the same or a different species; ifthe cells bearing the foreign gene include cells of the animal'sgermline, the gene may be transmissible to the animal's offspring.

Genetics constructs and methodologies of the invention may be used tocreate animals which due to their genetic make up will develop diabetesand will exhibit symptoms of the disease in a predictable period oftime. In a preferred embodiment, the transgenic mammal of the inventionexhibits a 80% incidence of diabetes within 70±10 days; more preferably,a 90% incidence of diabetes within 60±5 days; most preferably, a 95%incidence of diabetes within 55±5 days; and even more preferably, a 97%incidence of diabetes within 50±5 days. The animals of the invention areused in assays to test the ability of a candidate protective ordeleterious diabetes-mediating protein, or a test compound to prevent,enhance, or slow the development of diabetes.

In one aspect of the invention, genetic constructs and methodologies ofthe invention are used to create animals having a transgene encoding acandidate protective or deleterious protein. Transgenic animals may beselected from a genetic background predisposed for development ofdiabetes, or may be double-transgenic animals which will developdiabetes predictable and in a short period of time. In this embodimentof the invention, the animals are used in assays to test the ability ofthe candidate protective protein to inhibit or reduce the incidence ofdisease onset. In a related aspect, the animals are used in assays totest the ability of a compound to induce expression of a protectiveprotein and thus to protect the animal from disease development.

In one aspect of the invention, genetics constructs and methodologies ofthe invention are used to create animals which due to their genetic makeup will develop diabetes within a predictable period of time. Forexample, in one embodiment, transgenic animals are created which expressa deleterious diabetes-mediated disease such as mortalin. The animals ofthe invention are used in assays to test compounds able to prevent ordelay the onset of diabetes. In one embodiment, it is preferable toinclude a deleterious protein gene within the transgenic animal in arelatively high copy number, in that increasing the copy number tends todecrease the time required for disease onset.

Further, adjustments can be made with respect to the use of specifictypes of enhanced promoters in order to elevate the levels of expressionwithout increasing copy numbers. Specific types of enhanced promotersare known which would provide enhanced expression to thediabetes-mediating protein transgene without increased copy numbers. Theenhanced promoters may operate constitutively or inducibly.

The invention also provides a means of creating animal models fordiabetes or diabetes related diseases. The transgenic animals of theinvention provide a way to develop and test potential therapies for thediabetes, and will eventually lead to cures for this disease.

IV. Assays for Screening for Drugs Capable of Effecting the Expressionof Diabetes-Mediating Proteins.

Assay methods provided by the invention are useful for screen compoundscapable of effecting the expression of a diabetes-mediating protein, andthus the development of diabetes in a mammal. One model for screeningdrugs capable of effecting the expression of one or morediabetes-mediating proteins is the administration of compounds suspectedof having beneficial effects (including antisense oligonucleotides) tocells in culture. Useful cells are RIN, transfected, or islet cells. Theeffects of the test compound on protein expression may then be assayedby 2D gel electrophoresis.

Another screening model is an in vivo method with the use of a mammal atrisk for development of diabetes. Briefly, a mammal with an increasedrisk for diabetes (e.g., diabetes-prone BB rat or NOD mouse) is exposedto a test compound, and the effect of exposure to the test compound onthe development of diabetes determined.

The development of diabetes may be monitored throughout thedevelopmental period by determining the expression of one or morediabetes-mediating proteins and comparing the time of disease onset withexpression and timing in the absence of disease development. Determiningthe expression of one or more diabetes-mediating proteins includes thediabetes-mediating protein itself, a post-translational modificationproduct, and/or diabetes-mediating protein degradation product. In oneembodiment, activation of a diabetes-mediating protein is determined bymeasuring the level of the diabetes-mediating protein expression in atest sample. A suitable test sample includes a body fluid, such asblood, urine, or cerebrospinal fluid, or fluid derived from it, such asplasma or serum. In a specific embodiment, the level of proteinexpression in a test sample is measured by Western blot analysis. Theproteins present in a sample are fractionated by gel electrophoresis,transferred to a membrane, and probed with labeled antibodies specificfor the protein(s). In another specific embodiment, the level ofdiabetes-mediating protein expression is measured by Northern blotanalysis. Polyadenylated [poly(A)+] mRNA is isolated from a test sample.The mRNA is fractionated by electrophoresis and transferred to amembrane. The membrane is probed with labeled cDNA. In anotherembodiment, protein expression is measured by quantitative PCR appliedto expressed mRNA.

In a more specific embodiment, a mammal capable of developing diabetesis one selected from a strain of mammals which have been bred for anincreased incidence of diabetes. Preferable, the mammal is selected froma strain which exhibits a 75% chance of developing diabetes within 69±25days. More preferably, the mammal is a transgenic mammal of engineeredto exhibit a high incidence of diabetes within a predictable period oftime. In specific embodiments, the transgenic mammal exhibits a 80%incidence of diabetes within 70±10 days; more preferably, a 90%incidence of diabetes within 60±5 days; most preferably, a 95% incidenceof diabetes within 55±5 days; and even more preferably, a 97% incidenceof diabetes within 50±5 days.

In yet another aspect, the invention provides for methods foridentifying compounds capable of suppressing or reducing the expressionof an endogenous deleterious protein, as well as methods for preventingand/or treating diabetes by administering a therapeutically effectiveamong of a compound capable of suppressing or reducing the expression ofan endogenous deleterious protein.

The diabetes-mediating proteins of the invention are also useful toscreen reagents that modulate diabetes-mediating protein activity.Accordingly, in one aspect, the invention features methods foridentifying a reagent which modulates diabetes-mediating proteinactivity, by incubating a cell expressing a diabetes mediating proteinwith the test reagent and measuring the effect of the test reagent ondiabetes-mediating protein synthesis, phosphorylation, function, oractivity. When activation of a diabetes-mediating protein is viaphosphorylation, the test reagent is incubated with thediabetes-mediating protein and with either gamma-[labeled-ATP or[³⁵S]-methionine, and the rate of phosphorylation determined. In anotherembodiment, the test reagent is incubated with a cell transfected withan diabetes-mediating protein polynucleotide expression vector, and theeffect of the test reagent on diabetes-mediating protein transcriptionis measured by Northern blot analysis. In a further embodiment, theeffect of the test reagent on diabetes-mediating protein synthesis ismeasured by Western blot analysis using an antibody to thediabetesmediating protein In still another embodiment, the effect of areagent on diabetes-mediating protein activity is measured by incubatingdiabetes-mediating protein with the test reagent, [³²]P-ATP, and asubstrate in the diabetes-mediating protein pathway. All experimentswould be compared against a normal labeling of cells with[³⁵S]-methionine to determine modulation of protein expression. The rateof substrate phosphorylation is determined by methods known in the art.

The term modulation of diabetes-mediating protein activity includesagonists and antagonists. The invention is particularly useful forscreening reagents that inhibit deleterious protein activity. Suchreagents are useful for the treatment or prevention of diabetes.

V. Therapeutic Applications

The invention provides methods for preventing and/or treating diabetesin a mammal by administering a therapeutically effective amount of aprotective diabetes-mediating protein. Preferably the mammal is a humansubject at risk for diabetes.

Drug screening using identified diabetes-mediating proteins and relateddiabetes therapeutic agents. In a drug-screening assay of the invention,identified protective or deleterious diabetesmediating proteins are usedto identify test compounds capable of effecting their expression. Testcompounds so identified are candidate therapeutic agents for preventing,ameliorating, or delaying the onset of diabetes in a subject at risk.

A test therapeutic compound which effects the expression of adiabetes-mediating proteins can be, but is not limited to, at least oneselected from a nucleic acid, a compound, a protein, an element, alipid, an antibody, a saccharide, an isotope, a carbohydrate, an imagingagent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, andantibody or fragment thereof, or any combination thereof, which can bedetectably labeled as for labeling antibodies, as described herein. Suchlabels include, but are not limited to, enzymatic labels, radioisotopeor radioactive compounds or elements, fluorescent compounds or metals,chemiluminescent compounds and bioluminescent compounds.

A therapeutic compound is identified in the drug screening assay of theinvention through its ability to induce or enhance the expression of aprotective protein, such that disease onset is prevented or delayed in asubject at risk for the development of diabetes. A candidate therapeuticcompound is also identified by its ability to prevent or decrease theexpression of a deleterious protein, such that disease onset isprevented or delayed in a subject at risk for the development ofdiabetes.

A therapeutic nucleic acid as a therapeutic compound can have, but isnot limited to, at least one of the following therapeutic effects on atarget cell: inhibiting transcription of a deleterious protein DNAsequence; inhibiting translation of a deleterious protein RNA sequence;inhibiting reverse transcription of an RNA or DNA sequence correspondingto a deleterious protein; inhibiting a post-translational modificationof a protein; inducing transcription of a DNA sequence corresponding toa protective protein; inducing translation of an RNA sequencecorresponding to a protective protein; inducing reverse transcription ofan RNA or DNA sequence corresponding to a protective protein;translation of the nucleic acid as a protein or enzyme; andincorporating the nucleic acid into a chromosome of a target cell forconstitutive or transient expression of the therapeutic nucleic acid.

Therapeutic effects of therapeutic nucleic acids can include, but arenot limited to: turning off a defective gene or processing theexpression thereof, such as antisense RNA or DNA; inhibiting viralreplication or synthesis; gene therapy as expressing a heterologousnucleic acid encoding a therapeutic protein or correcting a defectiveprotein; modifying a defective or underexpression of an RNA such as anhnRNA, an mRNA, a tRNA, or an rRNA; encoding a drug or prodrug, or anenzyme that generates a compound as a drug or prodrug in pathological ornormal cells expressing the diabetes-mediating protein or peptide; andany other known therapeutic effects.

Also included in the invention is gene therapy by providing apolynucleotide encoding a protective diabetes-mediating protein.

The invention further includes a method for preventing diabetes byadministering an effective amount of a polynucleotide which inhibits thein vivo expression of a deleterious diabetes-mediating protein.

In the therapeutic method of the invention, a therapeutic compound isadministered to a human patient chronically or acutely. Optionally, aprotective protein is administered chronically in combination with aneffective amount of a compound that acts on a different pathway than thetherapeutic compound. The therapeutic method of the invention can becombined with other treatments for diabetes or with methods for themanagement of diabetes.

Therapeutic formulations of the therapeutic compound for treating orpreventing diabetes are prepared for storage by mixing the compoundhaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, 16th edition, Oslo, A., Ed., 1980), in the formof lyophilized cake or aqueous solutions. Acceptable carriers,excipients, or stabilizers are non-toxic to recipients at the dosagesand concentrations employed, and include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counter ions such as sodium; and/or non-ionicsurfactants such as Tween, Pluronics, or polyethylene glycol (PEG). Thecompound is also suitably linked to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The amount ofcarrier used in a formulation may range from about 1 to 99%, preferablyfrom about 80 to 99%, optimally between 90 and 99% by weight.

The therapeutic compound to be used for in vivo administration must besterile. This is readily accomplished by methods known in the art, forexample, by filtration through sterile filtration membranes, prior to orfollowing lyophilization and reconstitution. The therapeutic compoundordinarily will be stored in lyophilized form or in solution.

Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceableby a hypodermic injection needle.

The therapeutic compound administration is in a chronic fashion using,for example, one of the following routes: injection or infusion byintravenous, intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes, orally or usingsustained-release systems as noted below. The therapeutic compound isadministered continuously by infusion or by periodic bolus injection ifthe clearance rate is sufficiently slow, or by administration into theblood stream or lymph. The preferred administration mode is targeted tothe tissue of interest (p cell or pancreatic cells) so as to direct themolecule to the source and minimize side effects of the compound.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al. J Biomed Mater. Res. 15:167–277 (1981) andLanger Chem. Tech. 12:98–105 (1982), or poly(vinyl alcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. Biopolymers22:547–556 (1983)), non-degradable ethylene-vinyl acetate (Larger et al.supra (1981)), degradable lactic acid-glycolic acid copolymers such asthe Lupron Depot™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

The therapeutic compound also may be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interracialpolymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-[methylmethacylate] microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nanoparticles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease molecules for shorter time periods. When encapsulated moleculesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved, e.g., using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release compositions also include liposomally entrappedtherapeutic compound(s). Liposomes containing therapeutic compound(s)are prepared by methods known per se: DE 3,218,121; Epstein et al. Proc.Natl. Acad. Sci. USA 82:3688–3692 (1985); Hwang et al. Proc. Natl. Acad.Sci. USA 77:4030–4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83–118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomesare of the small (about 200–800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol % cholesterol, the selectedproportion being adjusted for the optimal agonist therapy. A specificexample of a suitable sustained-release formulation is in EP 647,449.

An effective amount of therapeutic compound(s) to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the clinician to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect.

If two therapeutic compounds are administered together, they need not beadministered by the same route, nor in the same formulation. However,they can be combined into one formulation as desired. Both therapeuticcompounds can be administered to the patient, each in effective amounts,or each in amounts that are sub-optimal but when combined are effective.In one embodiment, the administration of both therapeutic compounds isby injection using, e.g., intravenous or subcutaneous means, dependingon the type of protein employed. Typically, the clinician willadminister the therapeutic compound(s) until a dosage is reached thatachieves the desired effect for treatment or prevention of diabetes. Forexample, the amount would be one which ameliorates symptoms of diabetesand restores normoglycemia. The progress of this therapy is easilymonitored by conventional assays.

In specific embodiments of this aspect of the invention, the therapeuticcompound is a protective diabetes-mediating gene encodes gal-3, and/or apost-translational modification product of gal-3. A typical daily dosageof a therapeutic compound used alone might range from about 1 μg/kg toup to 100 mg/kg of patient body weight or more per day, depending on thefactors mentioned above, preferably about 10 μg/kg/day to 50 mg/kg/day.

Gene therapy. The present invention also provides gene therapy for thetreatment of diabetes and diabetes-related disorders, which are improvedor ameliorated by a protective polypeptide. Such therapy would achieveits therapeutic effect by introduction of the protective polynucleotideinto insulin-producing cells. Delivery of a protective polynucleotidecan be achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Especially preferred fortherapeutic delivery of sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector for invitro cell transformation is a derivative of a murine or avianretrovirus adenovirus. The advantage of adenovirus transduction comparedto other transfection methods is the high transfection affectivity andthe ability to transfect whole islets. Furthermore, the level ofexpression can be adjusted by the virus concentration andtransduction-time used. Even though the adenovirus-mediated expressionis transient, the expression in islets is stable for at least severalweeks (Becker et al. J. Biol. Chem. 269:21234 (1994); Korbutt et al.Transplantation Proc. 27:3414 (1995)).

For stable integration of a transgene into a mammal, a retroviral vectoris preferred. Examples of retroviral vectors in which a single foreigngene can be inserted include, but are not limited to: Moloney murineleukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murinemammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Preferably,when the subject is a human, a vector such as the gibbon ape leukemiavirus (GaLV) is utilized. A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a diabetes-mediating proteinsequence of interest into the viral vector, along with another genewhich encodes the ligand for a receptor on a specific target cell, forexample, the vector is now target specific. Retroviral vectors can bemade target specific by attaching, for example, a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibodyto target the retroviral vector. Those of skill in the art will know of,or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome or attached to a viral envelope to allow target specific deliveryof the retroviral vector containing the protective polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include, but are not limited to ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for protective polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LW), which range in size from0.2–4.0 μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al. Trends Biochem. Sci. 6:77(1981)). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Manning et al. Biotechniques 6:682 (1988)).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with sterols, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidyl-glycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipid,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidyl-glycerols, where the lipid moiety contains from 14–18carbon atoms, particularly from 16–18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine, dipaimitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

Due to the biological activity of therapeutic compounds in enhancingsynthesis of secondary therapeutic compounds, there are a variety ofapplications using the polypeptide or polynucleotide of the invention,including application to disorders related to the expression of suchsecondary proteins.

The invention provides methods for treatment of diabetes anddiabetes-related disorders, which are improved or ameliorated bymodulation of deleterious diabetes-mediating gene expression oractivity. In one specific embodiment of this aspect of the invention,the deleterious diabetes-mediating gene encodes mortalin. The term“modulate” envisions the suppression of expression of mortalin.

Where suppression of a deleterious protein expression is desirable, forexample, suppression of mortalin, nucleic acid sequences that interferewith mortalin expression at the translational level can be used. Thisapproach utilizes, for example, antisense nucleic acid, ribozymes, ortriplex agents to block transcription or translation of a specificmortalin mRNA, either by masking that mRNA with an antisense nucleicacid or triplex agent, or by cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub ScientificAmerican 262:40 (1990)). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target mortalin-producing cell.

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher et al. Antisense Res.and Dev. 1:227 (1991); Helene Anticancer Drug Design, 6:569 (1991)).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech J. Amer. Med Assn. 260:3030 (1988). A major advantage ofthis approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff Nature 334:585 (1988)) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11–18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

Blocking mortalin action either with anti-mortalin antibodies or with amortalin antisense polynucleotide may be useful for slowing orameliorating diabetes. The above described method for delivering aprotective polynucleotide are fully applicable to delivery of anmortalin antagonist for specific blocking of mortalin expression and/oractivity when desirable. A mortalin antagonist can be a mortalinantibody, an antisense polynucleotide sequence, or a compound whichsuppresses or inhibits the expression of mortalin.

Identification and Characterization of Diabetes-Mediating Proteins

High resolution two-dimensional (2D) gel electrophoresis may be used toseparate approximately 2500 protein spots, each spot corresponding to aprotein according to molecular weight and pI. A computerized 2-D geldatabase of 1373 WF rat islet proteins has recently been reported(Andersen et al. Diabetes 44:400407 (1995)) with a qualitativereproducibility of 81% (FIGS. 1A–1B). IL-1β has been shown to inducechanges in the expression of 82 protein in BB-DP islet cells incubatedin vitro. in vitro incubation of BB-DP islets taken from animals afterthe onset of diabetes shows that 33 of the 82 proteins determined torespond to IL-1β show significant changes in expression at the onset ofdiabetes.

Based on the results of 2D gel analysis and peptide sequencing or massspectroscopy analysis, several candidate diabetes-mediating proteinshave been identified (Table 1) and cloned from rat and human isletcells, including iNOS, mortalin, and galectin-3 (Madsen et al. inInsulin secretion and pancreatic B cell research, P. R. Flatt & S.Lenzen, eds., Smith-Gordon, USA, pp. 61–68 (1994)). Manganese superoxidedismustase (MnSOD) has been transfected into β-cells and whole islets ofLangerhans with the use of adenovirus mediated gene transfer andexpression. Transfection include the generation of stable or transientlytransfected cells.

Following transfection, subcloning, and establishment of stable RINclones expressing the coding region or antisense-fractions of theprotein of interest under the control of a CMV or insulin promoter, thediabetes-mediating protein of interest is functionally characterized.Preliminary results show a 100% transduction in RIN cells and up to 70%in isolated islets. Initially, the increased or decreased expression ofthe protein in the transfected cells is characterized in vitro bymeasurement of NO production, insulin secretion, and/or cytotoxicity.Furthermore, cell cycle and mitochondrial activity may be determined byFACS analysis (Mandrup-Poulsen et al. Diabetes/Metabolism Reviews9:295–309 (1993)) and semiquantitative analysis of gene expressioncharacterized by multiplex PCR (Nerup et al. Anales Espanoles Pediatria58:4041 (1994)). Additionally, secondary cellular effects of the over-or under-expression of the protein as well as posttranslationalmodification may be characterized by 2D-gel electrophoresis of thetransfected cells. This analysis allows a distinction to be made betweenprimary and secondary effects in the IL-1β induced changes in proteinexpression pattern.

To establish IL-1β induced changes in islet cells, NO production andinsulin release were measured in BB-DP islets of Langerhans in vitroafter IL-1β exposure and high resolution two-dimensional gelelectrophoresis to detect protein changes resulting from IL-1 exposure(Example 2). NO production and insulin release in control islets wassimilar to that observed in WF islets. IL-1β inhibited NO production4.4-fold and insulin release 3.4-fold. 2D gel analysis showed that IL-1βchanged the expression of 82 proteins-22 proteins were up-regulated and60 down regulated.

High resolution 2DGE was also used to detect protein changes in BB-DPislet syngrafts transplanted into 30 day old BB-DP rats (Example 3). InBB-DP islet syngrafts from newly onset diabetic BB-DP rats, but not inWF islet syngrafts, 15 of these 82 proteins were found to change levelof expression (Example 4).

The IL-1β induced changes in protein expression in vitro were comparedto protein changes in the process of disease occurrence in BB-DP isletsyngrafts (Example 4) or in BB-DP islet allograft rejection (Example 6).In BB-DP islet syngrafts from newly onset diabetic BB-DP rats, but notin WF islet syngrafts 15 of these 82 proteins were found to change levelof expression.

Proteins exhibiting altered expression with diabetes onset in syngraftedislets were further characterized (Example 5). One of the alteredprotein spots was identified by amino acid sequencing to have highhomology to the heat shock 70 protein (mortalin), which has beendemonstrated to be involved in cellular mortality and apoptosisfollowing translocation of this constitutively expressed protein from aperinuclear to the cytoplasmic region (Wadhwa et al. J. Biol. Chem.268:6615 and 268:22239 (1993)). Based on the amino acid sequence,mortalin cDNA was cloned from rat and human islet for furthercharacterization of its involvement in diabetes development.Over-expression of mortalin in rat insulinoma (RIN) β cells under a CMVpromoter was lethal to β cells.

Another diabetes-mediating protein was identified as galectin-3.Galectin-3 (gal-3) was identified in 2D gels as spots iS (phosphorylatedtwice), 16 (phosphorylated once), and 19 (nonphosphoryolated) (Andersenet al. Diabetes 44:400–407 (1995)) (Example 5). Gal-3 is a proteininvolved in islet development and inhibition of apoptosis. Using thenucleotide sequence, gal-3 was cloned from rat and human islets,subcloned and expressed in RIN cells after selection for stable clones.RIN cells expressing gal-3 exhibited an increased metabolic activity andproliferative rate, and became more resistant to the negative effect ofcytokines. The in vivo effect of gal-3 expression was studied bytransplanting 200 neonatal islets to 30 day old diabetes proneBio-Breeding rats (BB-DP) (Example 8).

Further work using the NIGMS (National Institute of General MedicalScience) human/rat somatic cell hybrid mapping panel # 2 and primersfrom intron 2 of the gal-3 gene, has mapped gal-3 to chromosome 14.Using the same primers, a PI clone was obtained from GenomeSystems, Inc.to use for FISH. Initial FISH results obtained with 48 measurementsshowed 6.2% localized to the 14q13 region, 54.2% to the 14q21 region,and 39.6% to the 14q22 region, which indicate a distal location in the14q21.3 region.

Inducible nitric oxide synthase (iNOS) was cloned (Karlsen et al.Diabetes 44:753 (1995)). The iNOS gene was mapped to mouse chromosome11, which is in the middle of the idd4 region; a region identified as adiabetes-linked region in the diabetes prone NOD mouse (Gerling et al.Mammalian Genome 5:318 (1994)). When expressed as a recombinant proteinin fibroblasts, it was found that the recombinant iNOS was enzymaticallyactive.

Manganese superoxide dismutase (MnSOD) was identified as a downregulated protein by 2D gel analysis. The expression of MnSOD wasfurther characterized in β and islet cells in vitro and in vivofollowing adenovirus transduction and transplantation. Mitochondrialisocitate dehydrogenase was identified as a diabetes-mediating protein.

Changes in protein expression associated with allograft rejection werealso determined. Neonatal BB-DP islet cells transplanted into. 30 dayold WK rats were retrieved after 12 days, and protein expressiondetermined (Example 6). It was found that BB-DP islet allografts in WKrats but not in WF islet syngrafts 9 of 82 proteins were found to changelevel of expression.

Both the syn-and allografted BB-DP islets were compared to non-graftedBB-DP islets with regard to the proteins found to change expressionlevels during IL-1 exposure of the BB-DP islets in vitro. To control forprotein-changes induced by the grafting procedure, 200 neonatal WFislets were grafted to 30 day old WF rats. Grafts were retrieved 48 daysafter transplantation corresponding to the mean time of onset ofdiabetes in the colony and in the syngrafted BB-DP rats. The grafts wereprocessed and analyzed as described for comparison with WF controlislets with regard to the 105 proteins previously found to be changedduring IL-1 incubation. This was done to identify protein changesinducible both by IL-1 and islet syngrafting as well as to identifyrejection-specific proteins.

To further determine a potential role of these proteins in the diabetespathogenesis, and to identify therapeutic compounds and compounds whichinduce the expression of therapeutic compounds, transgenic animalscarrying the gene encoding the candidate protein and able to express thecandidate protein under tissue-specific promoters are generated. Thetransgenic animals of the invention express candidate proteins underspecific promoters such as the insulin, amylin, CMV, or HLA promoters.

Initially, a study was conducted to determine the optimal conditions foradenoviral-mediated gene transfer to the islets of Langerhans in theabsence of vector-induced toxicity. As described in Example X,adenoviral mediated transduction of islets resulted in dose-dependentefficient gene transfer with stable transgene expression in the absenceof toxicity.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the chimeric genes, transgenic mice and assays of thepresent invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g. amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Materials and Methods

Reagents. Ketamin was purchased from Park-Davis (Barcelona, Spain),xylazin from Bayer (Leverkusen, Germany), and Temgesic® from Reckitt andColemann (Hull, UK). RPMI 1640, Hanks' balanced salt solution (HBSS),and DMEM were purchased from Gibco, Paisley, Scotland. RPMI 1640contained 11 mmol D-glucose, and was supplemented with 20 mM HEPESbuffer, 100,000 IU/I penicillin and 100 mg/1 streptomycin. Authenticrecombinant human IL-1β, was provided by Novo Nordisk Ltd. (Bagsvaerd,Denmark) having a specific activity of 400 U/ng.

Other reagents used: 2-mercaptoethanol, bovine serum albumin (BSA), TrisHCl, Tris base, glycine, (Sigma, St. Louis, USA); trichloracetic acid(TCA), phosphoric acid, NaOH, glycerol, n-butanol, bromophenol blue,sodium nitroprusside (SNP), H₃PO₄ and NaNO₂ (Merck, Darmstadt, Germany);filters (HAWP 0.25 mm pore size) (Millipore, Boston, USA); RNAse A,DNAse I (Worthington, Freehold, N.J., USA); [³⁵S]-methionine (SJ 204,specific activity:>1.000 Ci/mmol, containing 0.1% 2-mercaptoethanol),Amplify® (Amersham International, Amersham, UK); urea (ultra pure)(Schwarz/Mann, Cambridge, Mass., USA); acrylamide, bisacrylamide, TEMED,ammonium persulphate (BioRad, Richmond, Calif., USA); ampholytes: pH5–7, pH 3.5–10, pH 7–9, pH 8–9.5 (Pharmacia, Uppsala, Sweden); NonidetP-40 (BDH, Poole, UK); ampholytes: pH 5–7 and sodium dodecyl sulphate(Serve, Heidelberg, Germany); agarose (Litex, Copenhagen, Denmark);ethanol (absolute 96%) (Danish Distillers, Aalborg, Denmark); methanol(Prolabo, Brione Le Blanc, France); acetic acid (technical quality, 99%glacial) (Bie & Bemtsen, Arhus, Denmark) and X-ray film (Curix RP-2)(AGFA).

Animals. Thirty (28–32) day old BB/Wor/Mol-BB2 (BB-DP), Wistar Furth(WF) and Wistar Kyoto (WK) rats were purchased from Møllegården, Li.Skensved, Denmark. At Møllegården and in the animal facilities at theSteno Diabetes Center/Hagedorn Research Institute (Gentofte, Denmark)the BB-DP rats were housed separately in a specific pathogen-freeenvironment. All rats were housed under controlled conditions of light(light on from 6:00 am to 6:00 pm), humidity (60–70%) and temperature(20–22° C.) from five days prior to transplantation until sacrifice. Therats were offered a standard rat chow (Altromin, Chr. Petersen A/S,Ringsted, Denmark) and free access to tap water. Four to five day-oldBB-DP and WF rats were picked up at Møllegråden in the morning on theday of islet isolation, and transported in animal transport boxes.

Transplantation procedure and graft retrieval The neonatal islets weretransplanted under the kidney capsule of 28–32 day-old recipient ratsunder sterile conditions as previously described (Korsgren et al. J.Clin. Invest. 75:509–514 (1990)). Since diabetes incidence is similar inmale and female BB-DP rats (Pries et al. Frontiers in Diabetes Research8:19–24 (Shafrir, E., ed.), Smith-Gordon, London (1991) both sexes wereequally used. To reduce a possible risk of graft rejection due to anygender-related incompatibility, male islets were transplanted to malerecipients and female islets to female recipients (Steiner et al.Electrophoresis 16:1969–1976 (1995)). Two-hundred (range: 187–215)islets were handpicked using a 10 ml Transpferpettor (Brand, Germany)pipette. The islets were allowed to sediment, and as much supernatant aspossible was removed prior to the transplantation of a volume of 1–2 ml.The recipient rats were weighed, and blood glucose (BG) was measured.The rats were then anesthetized with ketamin (8.75 mg/100 g) and xylazin(0.7 mg/100 g) ip. A small incision was made over the left kidney, thekidney was lifted forward, and a small incision using a knife was made.A pocket using a blunt instrument was gently made in the capsule. Theislets were placed under the capsule towards the lower kidney pole. Themuscle and skin were sutured, the rats allowed to recover in a cage inthe operating facility and given Temgesic (0.01 mg) sc in the neckbefore returning to the animal facility. The rats were given Temgesictwice daily for 3 days post transplantation. Blood glucose (BG) wasmeasured every third day. The rats were killed by cervical dislocationand immediately afterwards the grafted kidneys were removed. The graftswere carefully dissected from the kidneys and capsules under amicroscope and placed in HBSS.

Graft and islet labeling. The grafts were labeled immediately afterretrieval. Cultured islets were labeled after 24 h of incubation with orwithout IL-1. Grafts and islets were washed twice in HBSS and labeledfor 4 h at 37° C. in 200 ml methionine-free Dulbecco's modified Eagle'smedium (DMEM) with 10% NHS dialyzed for amino acids, and either 330 mCi[35S]-methionine for the grafts or 200 mCi [35S]-methionine for theislets. To eliminate 2-mercaptoethanol, [35S]-methionine wasfreeze-dried for at least 4 h before labeling. After labeling, thegrafts and islets were washed three times in HBSS, the supernatant wasremoved and the tissue immediately frozen at −80° C.

Sample preparation. The frozen grafts were crushed in a mortar. Thegrafts and islets were resuspended in 100 ml DNAse I/RNAse A solutionand lysed by freeze-thawing twice. After the second thawing, the sampleswere left on ice for 30 min for the digestion of nucleic acids and thenfreeze dried overnight. The samples were dissolved by shaking in 120 mllysis buffer (8.5 M urea, 2% Nonidet P-40, 5% 2-mercaptoethanol and 2%ampholytes, pH range 7–9) for a minimum of 4 h.

Determination of [35S]-methionine incorporation. The amount of[35S]methionine incorporation was quantitated by adding 10 mg BSA (0.2mg/ml H₂O) as a protein-carrier to 5 ml of a 1:10 dilution of eachsample in duplicate, followed by 0.5 ml of 10% TCA. This was left toprecipitate for 30 min at 4° C. before being filtered through 0.25 mmHAWP filters. The filters were dried and placed into scintillationliquid for counting.

2-D gel electrophoresis. The procedure has been described earlier(O'Farrell et al Cell 12:1133–1142 (1977)). Briefly, first dimensiongels contained 4% acrylamide, 0.25% bisacrylamide and ampholytes. Equalnumbers of counts (106 cpm) of each sample were applied to the gels. Incase of lower amounts of radioactivity it was necessary to regulate theexposure time of the gel so that comparable total optical densities wereobtained. The samples were analyzed on both isoelectric focusing (IEF;pH 3.5–7) and non-equilibrium pH-gradient electrophoresis (NEPHGE; pH6.5–10.5) gels. Second dimension gels contained 12.5% acrylamide and0.063% bisacrylamide and were run overnight. After electrophoresis, thegels were fixed and treated for fluorography with Amplify® before beingdried. The gels were placed in contact with X-ray films and exposed at−70° C. for 3 to 40 days. Each gel was exposed for at least 3 timeperiods to compensate for the lack of dynamic range of X-ray films.

Determination of MW and pI. Molecular weights of the proteins weredetermined by comparison with standard gels. The pIs for the individualproteins on the gels were determined by the use of pI calibration kits.Landmark proteins were identified on gels by one or several of thefollowing techniques: immunoblotting, immunoprecipitation, microsequencing or peptide mapping.

Computer analysis of fluorographs. Computer analysis was performed usingthe BioImage® program (version 6.1) on a Sunsparc workstation. First,the fluorographs were scanned and spots identified and quantitated bythe BioImage® program. Next, anchor points were placed on the gel (samespot in each gel was assigned the same anchor point), and the computerwas asked to match the gels. After computer matching, manual editing wasperformed to ensure identification, correct matching of computer foundspots and quantitation of spots not found initially by the computerprogram. Finally, data were extracted for calculations in the QuatroPro® spreadsheet (Borland version 4.0). To avoid the presence ofduplicate spots in the IEF and NEPHGE subgroups, overlapping spots ineither the basic part of IEF gels or in the acidic part of NEPHGE gelswere omitted from analysis.

Statistical analysis. Student's t test was applied and P<0.01 was chosenas level of significance.

Example 2 In Vitro Nitrite and Insulin Determinations

To investigate IL-1 induced protein changes in BB-DP islets, 150neonatal BB-DP rats islets were incubated for 24 hours with or without150 pg/ml of IL-1, after which the medium was sampled for nitrite andinsulin measurements. Two dimensional gel analysis of the islets wasperformed as described below, and fluorographs of the islets wereanalyzed on computer (see below, n=3 for each group) and compared withthe previously established protein database of IL-1 exposed WF ratislets.

Islet isolation and culture. Islets from pancreata of four to five dayold inbred BB-DP and WF rats were isolated after collagenase digestionas described by Brunstedt et al. in Methods in Diabetes Research, Vol.1, Wiley & Sons, New York, pp. 254–288 (1984), specifically incorporatedherein by reference. After 4 days of preculture in RPMI 1640+10% fetalcalf serum, islets were cultured as follows: 150 BB-DP islets wereincubated for 24 h in 37° C. humidified atmospheric air in 300 ml RPMI1640+0.5% normal human serum (NHS) with or without the addition of 150pa/ml IL-18. In separate series of experiments, 200 BB-DP or WF isletswere incubated for 24 h in 300 ml RPMI 1640+0.5% NHS before grafting.

Nitrite and insulin measurements. Nitrite was measured by Griessreagents as previously described (Green et al. Anal. Biochem.126:131–138 (1982)). The detection limit of the assay was 1 mmol/l,corresponding to 2 pmol/islet. The nitrite level of the correspondingmedium without islets was subtracted if the value of the islet-freemedium was above the detection limit. The experiments were run in thesame assay. Intra- and interassay coefficients of variation calculatedfrom 3 points on the standard curve were: 1 mmol/l: 1.6%, 16.3%; 10mmol/l: 1.6%, 15.3%; 25 mmol/l: 1.5, 17.0%. Insulin was measured by RIA(Yang et al. Proc. Natl. Acad. Sci. 93:6737–6742 (1996)). The detectionlimit was 35 fmol/ml. Intra- and interassay coefficients of variationbetween 3 known controls were A: 5.4%, 12.5%; B: 3.6%, 10.2%; C, 3.1%,9.4%.

Results. To ensure comparability in terms of function (accumulatedinsulin release) and response to IL-1 between BB-DP islets and the WFislets previously used for 2-D gel analysis (Andersen et al. supra(1995)), NO production and insulin release were measured after 24 hoursof incubation with or without IL-1. The basal insulin release and NOproduction were similar to data from WF islets (basal insulin release(WF islets) 3.3±0.5 versus (BB-DP islets) 2.0±1.1 pmol*islet-1*24h−1,basal NO production <2 pmol islet-1* 24h−1, for both BB-DP and WFislets. IL-1 inhibited accumulated islet insulin release by 3.4 fold andincreased NO production by 4.4 fold, similarly to results obtained usingneonatal WF rat islets (IL-1 induced NO production: (WF islets)9,29±1,21 versus (BB-DP islets) 8.91±1.05 pmol*islet-1*24 h−1 andinsulin release: (WF islets) 1,4±0,3 versus (BB-DP islets) 0,6±0.6pmol*islet-1*24 h−1,18,19).

Analysis of fluorographs from BB-DP rat islets±IL-1. In a computer basedanalysis of two dimensional gels of neonatal BB-DP rat islet homogenatesa total of 1815 (1275 IEF and 540 NEPHGE) proteins were found in 3 of 312,5% acrylamide gels. In neonatal BB-DP rat islets incubated with IL-1for 24 hours, 1721 (1171 IEF and 550 NEPHGE) were found in 3 of 3 gels.IL-1 was found to significantly change the expression level of 82proteins compared to control islets (p<0.01); 60 proteins were decreasedand 22 increased in expression level (p<0.01) (Table 1).

Example 3 Animal Transplantation Model

Experiment 1. Initially, to create a synchronized model for theinvestigation of the cellular and molecular events occurring duringdevelopment of IDDM, 200 neonatal BB-DP rat islets were isolated andtransplanted under the kidney capsule of 30 day old BB-DP rats. Bloodglucose was measured every third day, and the incidence of diabetesdetermined.

The rats were sacrificed 7, 12, 23, 37, 48, and 173 days aftertransplantation or at onset of diabetes (n=6 in each group). Grafts weredouble stained for insulin and one of the following MHC class I, II,αβ-TCR, CD4, CD8, or ED1. Staining was expressed as percentage of totalgraft area (insulin) or number of stained cells per mm² The host isletsin situ were stained in the same way and expressed as percent inflamedislets of the total number of islet cells. Statistical analysis wasconducted with Spearman Rank Correlation and Mann-Witney.

Results. The incidence of diabetes (75%) or day of onset of diabetes(69±25 days) did no differ significantly from non-transplanted BB-DPrats. A 2–3 fold increase in the number of infiltrating cells in bothgraft and islets in situ was seen days 12 and 37. Forty-eight days aftertransplantation, the number of cells increased in both graft and isletsin situ for the rats developing diabetes (p<0.04) compared to day 37 inprediabetic and non-diabetic rats at day 48, respectively. Graft insulinstaining area was 80–90% in the nondiabetic and 30% of total graft areain the diabetic grafts with an inverse relationship between insulincontent and cellular infiltrate. For all cell types and at alltimepoints studied, a positive correlation was found between thepercentage of infiltrated islets in the host pancreatic slides and thenumber of infiltrating cells in the graft from the same animals(p=0.002−0.00001; r=0.4−0.7).

Experiment 2. 200 neonatal BB-DP rat islets were transplanted under thekidney capsule of 30 day old BB-DP rats. Allogeneic transplantation fromWistar Furth islets to Wistar Kyoto rats was similarly conducted as acontrol.

The grafted islets did not affect IDDM incidence or time of diseaseonset. On the day of diabetes onset (78±5 days) and on day 17 in thecontrol WK animals, the grafts were excised for immunohistochemistry and[³⁵S]-methionine labeling.

Results. Immunohistochemical examination of the grafts from diabetic BBrats demonstrated massive inflammation with MHC class I positive cells(1982/mm²±534), macrophages (661/mm²±406), T-helper cells(1331/mm²±321), cytotoxic T-cells (449/mm²±117), and insulin positivecells reduced to 33–66$ of the transplant. Allogeneic transplantcontrols showed fibrosis, less infiltration of mononuclear cells, MHCclass II positive cells (152/mm²), MHC class I positive cells (48/mm²),macrophages (172/mm²), T-helper cells (254/mm²), cytotoxic T-cells(42/mm²), and insulin content of 100% in the remaining cells.

Computerized analysis of 2D gels showed a greater than 2-fold increasein the expression of 18 of the 33 proteins altered in vitro by IL-1exposure. 14 were specific to the syngeneic transplants in the BB-DPanimals. 8 proteins specifically changed level of expression.

Example 4 Changes in Protein Expression Induced by Diabetes Onset

To investigate protein changes in BB-DP islet syngrafts during diseaseoccurrence, 200 neonatal BB-DP rat islets were grafted to 30 day oldBB-DP rats. Grafts were retrieved at onset of diabetes, defined as BG>14mmol/l for two consecutive days, n=5, for labeling, two dimensional gelelectrophoresis and computer based comparison with non-grafted BB-DPislets.

Analysis of fluorographs from syngeneic islet-grafts from newly onsetdiabetic BB-DP rats. In BB-DP islet syngrafts from newly onset diabeticBB-DP rats, computer analysis showed 1818 proteins (1259 IEF and 557NEPHGE) present in 5 of 5 gels. (These data are preliminary proteinspresent in both islets in vitro and grafts changed expression level (64were increased and 131 decreased in expression level) when compared tocontrol neonatal BB-DP rat islets (p<0.01, data not shown)). Seventy-oneof the eighty-two proteins that changed level of expression in BB-DP ratislets after IL-1 incubation in vitro were re-identified in the graft.Of the 71 re-identified proteins 33 significantly changed expressionlevels (4 up- and 29 down-regulated) in the islet syngrafts at onset ofdiabetes (Table 2).

Analysis of fluorographs from islet WF syngrafts. To control for changesin protein expression caused by the grafting procedure per se, WF isletsyngrafts were compared to the protein changes caused by IL-1 in WFneonatal islets in vitro. Of the 105 proteins that changed expressionlevel in WF islets incubated with IL-1 in vitro, 89 proteins werere-identified whereas 12 were absent in the WF islet grafts (n=3) whencompared to the WF control islets. Forty-two of the ninety-threere-identified proteins significantly changed level of expression (27decreased and 15 increased, p<0.01, Table 5). IL-1 induced proteins invitro specifically seen in islet-grafts during disease occurrence orislet-graft rejection. Proteins that were found to change level ofexpression in islets after IL-1 incubation in vitro and not found tosignificantly change level of expression in syngrafted WF islets weredenoted specific for disease occurrence if found in syngrafted BB-DPislets or specific for graft-rejection if found in allografted BB-DPislets to WK rats. In syngrafted BB-DP islets 28 proteins and inallografted BB-DP islets 29 proteins changed level of expression (datanot shown). Fourteen proteins specifically changed level of expressionin BB-DP syngrafts and 8 in BB-DP allografts in WK rats.

Example 5 Characterization of Diabetes-Mediated Proteins

Proteins exhibiting IL-1β-induction of synthesis were analyzed by massspectrometry and microsequencing as described below. Commerciallyavailable protein databases were searched for matches, includingSWISS-PROT, PIR, NIH, and GENEBANK.

Microsequencing. Protein spots identified by 2DGE as diabetes-mediatedproteins were further characterized by being digested with trypsin inthe gel, concentrated, HPLC separated, peak sequenced, and partialsequence compared to known sequences, according to known methods.

Results of microsequences are as follows:

-   Protein 22 (peak 22): A Q Y E E L I A N G (D) (M) (SEQ ID NO:5)    -   (peak 13):K K P L V Y D E G (K)(SEQ ID NO:6)-   Protein 25 (peak 15): L L E X T X X L X (SEQ ID NO:7)    -   (peak 16): P S L N S X E X (SEQ ID NO:8)-   Galectin-3 (peak 22): I E L X E I X (SEQ ID NO:9)

Direct sequencing of one protein (mw 68,700) yielded the followingpartial sequence: PEAIKGAVVGIDLG (SEQ ID NO:10) (Table 2: GR75). Thisprotein has high homology to 75 kD glucose regulated protein (GR75,Table 2) (PBP74, P66mot, mortalin); is a constitutive member of hsp70protein family, not heat-inducible; is ubiquitously expressed indifferent tissues; comprises a 46 residue leader peptide 75 kD processedto 66 kD; is found in mitochondria; is associated with cellularmortality and with antigen presentation.

One protein was identified as galectin-3, a 27 kD protein which ispresent in several tissues, and known to have a role as a pre-mRNAsplicing factor. The amino acid sequence of human gal-3 is shown in FIG.5 (SEQ ID NO:4).

Mass spectroscopy. In situ digestion is performed on at least one gelplug including at least one protein spot in at least one gel accordingto the present invention. Gels are prepared by a modification of themethod of Rosenfeld et al. Anal. Biochem. 203:173–179 (1992), asdescribed in Fey et al. Electrophoresis 18:1–12 (1997), both of whichreferences are herein specifically incorporated by reference. Briefly,gels are quickly stained and destained. The protein of interest isobtained by cutting a gel band containing the protein with a scalpel andstoring in eppendorf tubes with UHQ water at −20° C. The protein isdigested by washing the gel plug for at least 1 hour in 40%acetonitrile/60% digestion buffer until the coomassie stain is removed.This wash removes coomassie stain, gel buffers, SDS and salts. Ifnecessary the wash can be repeated. The gel plug is then dried in avacuum centrifuge for 20–30 min. until the plug shrinks and becomeswhite on the surface. Drying time depends on the size and thickness ofthe gel plug. Trypsin (or the enzyme being used is dissolved indigestion buffer and 5 mls added to the gel plug (depending on theamount of the protein in the gel to be analyzed (0.1 mg)). Additionaldigestion buffer is added until the gel plug is almost covered by bufferin the bottom of the tube, approximately 10 ml. The gel plug is thenincubated at 37° C. for 6 hours or overnight, then incubated with 70–100ml 60% acetonitrile/40% water for 2–6 hours to extract the peptides. Theextraction may be repeated to increase recovery. The extract is thenlyophilized and dissolved in 30% acetonitrile/2% TFA before analyzing byMALDI-MS.

FIGS. 6–48 provide the mass spectroscopic data obtain for the indicatedprotein of Tables 1 and 2.

Example 6 In Vivo Protein Expression During Allograft Rejection

To investigate protein changes in BB-DP islet allografts duringrejection, 200 neonatal BB-DP rat islets were grafted to 30 day old WKrats (n=3). Grafts were retrieved 12 days after transplantation at whichtime point substantial mononuclear infiltration is expected. The graftswere processed and analyzed as described above.

Analysis of fluorographs from BB-DP islet allografts in WK rats. Twelvedays after transplantation mononuclear cell infiltration was observed inthe grafts (18), and computer analysis of fluorographs from the BB-DPislet allografts sampled at this time-point showed 1714 (1064 IEF and650 NEPHGE) proteins present in 3 of 3 gels. The gels of the BB-DP isletallografts were compared to gels of neonatal BB-DP rat islets withregard to the 82 proteins that significantly changed level of expressionin the BB-DP islets after IL-1 incubation in vitro. Sixty-six of theeighty-two proteins were re-identified. Thirty-three of the sixty-sixre-identified proteins were found to change expression levels (28decreased and 5 increased, p<0.01).

Example 7 Establishment of Non-Toxicity of Adenoviral-Mediated GeneTransfer in Islet Cells

To determine the optimal conditions for adenoviral-mediated genetransfer to the islets of Langerhans in the absence of vector-inducedtoxicity, neonatal rat islets were transduced in groups of 25 intriplicate with an adenoviral vector β-galactosidase (AdβGal) at dosesof multiplicity of infection (moi) 0, 10, 100, and 1000 pfu per islet.Efficiency of gene transfer was determined by gross inspection andestimated by the percentage of, 6-galactosidase positive cells afterislet dispersion at 1, 4, 7, and 10 days post-transduction. Islettoxicity was assessed by measuring accumulated insulin levels at eachtime-point and by assessing insulin release in response to hyperglycemiaat 3 and 10 days.

Results. Efficient dose-dependent gene transfer to the islets wasdocumented at 1, 4, 7, and 10 days post-transduction. At day 1, 8.3%,34.1%, and 58.6% of cells in dispersed islets expressed transgene at moi10, 100, and 1,000, respectively. Transgene expression was stable forthe duration of the experiment. Insulin accumulation did not differbetween transduced and non-transduced islets at each time-point; andaccumulated insulin at 10 days expressed as nmol of insulin per isletwas 14.3±1.5 at a moi of 0, 15.0±2.6 at a moi of 10, 22.0±7.8 at a moiof 100, and 15.3±1.5 at a moi of 1,000.

Similarly, the insulin secretory response to glucose, obtained bydividing the insulin response to high glucose incubation by the insulinresponse to low glucose incubation was similar in transduced andnon-transduced islets at 3 days at all doses studied (mod 0; 12.7±4.1;moi 10, 14.9±7.9; moi 100, 15.7±0.7; and moi 1,000, 22.3±6.7). Theratios were similar in transduced and non-transduced cells at 10 dayspost-transduction.

Example 8 Expression of Galectin-3 in the Spontaneous Development ofIDDM

200 neonatal BB-DP rat islets were transplanted under the kidney capsuleof 30 day old BB-DP rats. Grafts retrieved day 7 after transplantation(prediabetic, n=6), at diabetes onset (n=6) or day 174 aftertransplantation (animals escaping diabetes, n=3) and IL-113 stimulatedand non-stimulated neonatal BB-DP islets were labeled with[35S]-methionine and prepared for high-resolution two-dimensional gelelectrophoresis.

Each sample was run on isoelectric focusing (IEF, pH 3.5–7) andnon-equilibrium pH-gradient electrophoresis (NEPHGE, pH 6.5–10.5).Fluorographs of the gel were analyzed as described above. Changes inexpression levels of proteins were considered significant at p valuesbelow 0.01 (Student's t-test).

Results. Neonatal BB-DP islets stimulated with IL-1β in vitro showedchanges in 82 proteins. 97–98% of these proteins were identified in allgrafts in all time points. Expression levels of graft proteins comparedwith non-stimulated normal BB-DP islets in vitro showed the followingchanges: Of the 82 proteins which exhibit increased expression whenneonatal islet cells are treated with IL-1β in vitro, 42 were increasedin the 7 day transplant islets, 31 were increased with diabetes onset,and 14 were increased in animals which did not develop diabetes. Of theproteins increased, 4 of them are only seen at onset of IDDM and not atthe timepoints detailed. At day 7, five proteins which do not changewith in vitro IL-1β exposure were observed to change, while 2 proteinswere observed to change at the onset of diabetes, and 3 in the animalswhich did not develop diabetes. Of the proteins observed to change invivo but not in vitro, I one of these is identified as IDDM specific.Gal-3 expression was significantly down-regulated at day 7 and at onsetof IDDM. In contract, gal-3 expression was increased in in vitro IL-1βstimulated islets and in grafts from animals which did not developdiabetes.

Example 9 Cytokine Induction of IL-1 Converting Enzyme (Ice), InducibleNitric Oxide Synthase (iNOS), and Apoptosis in Insulin-Producing Cells

The expression of ICE, iNOS, and induction of apotosis was investigated.Rat insulinoma and pluripotent cells lines RIN-SAH and MSL-G2 werecultured 20 hours with a mixture of cytokines (IL-1β, TNFoα, and IFNγ),RNA isolated, and multiplex PCR analysis (27 cycles) with primersagainst ICE, iNOS, and SP-1 (a general transcription factor used as ahousekeeping control gene for normalization) were formed. Results werenormalized to SP-1 mRNA expression, and calculated following gelelectrophoresis separation and PhosphorImager quantification.

Results. Neither ICE or iNOS expression were detected in control cells.A marked up-regulation to that of the level of the SP-1 gene product wasseen in both ICE and iNOS after 20 hours of cytokine exposure. In RINcells, ICE was produced at 98% and iNOS produced at 97% of SP-1. Whereasneither apoptosis or NOS production were detected in the control cells,cytokine-induced iNOS production was followed by a high increase inapoptosis-frequency and NO production. In RIN cells, accumulated nitritewas measured to be 19.7±0.7 μM after 3 days.

TABLE 1 ISLET CELL DIABETES MEDIATING PROTEINS WHICH INCREASE UPON IL-18STIMULATION IDENTIFIED BY 2DGE Database Gel Spot No. Gene ProteinAccession IEF 010 Unknown MWt: 120,500; pI: 7.27 IEF 011 EF2 ELONGATIONFACTOR 2 P05197 (EF-2) IEF 025 VAT VACUOLAR ATP P31408 SYNTHASE SUBUNITB, BRAIN ISOFORM (EC 3.6.1.34) IEF 028 MMLAMIN11 LAMIN B1 (mouse) D50080IEF 083 PYC PYRUVATE P52873 CARBOXYLASE PRECURSOR (EC 6.4.1.1) IEF 085PYC PYRUVATE P52873 CARBOXYLASE PRECURSOR (EC 6.4.1.1) IEF 115 UnknownMWt: 175,200; pI: 5.23 IEF 145 Unknown MWt: 133,100; pI: 6.29 IEF 173MVP MAJOR VAULT Q62667 PROTEIN IEF 186 HS74 HEAT SHOCK 70 KD Q61316PROTEIN AGP-2 (mouse) IEF 187 HS74 HEAT SHOCK 70 KD Q61316 PROTEIN AGP-2(mouse) IEF 189 Unknown MWt: 135,100; pI: 4.96 IEF 194 UBP1 UBIQUITINCARBOXYL- P45974 TERMINAL HYDROLASE T (EC 3.1.2.15) IEF 201 CALXCALNEXIN PRECURSOR P35565 IEF 210 Unknown MWt: 114,500; pI: 6.40 IEF 217Unknown MWt: 91,300; pI: 6.12 IEF 225 Unknown MWt: 83,300; pI: 5.89 IEF265 TERA TRANSITIONAL P46462 ENDOPLASMIC RETICULUM ATPASE (TER ATPASE)IEF 267 HS9B HEAT SHOCK PROTEIN P34058 HSP 90-BETA (HSP 84) IEF 276Unknown MWt: 107,500; pI: 4.34 IEF 279 Unknown MWt: 121,400; pI: 4.20IEF 285 Unknown MWt: 78,300; pI: 6.66 IEF 289 Unknown MWt: 73,900; pI:6.44 IEF 306 Unknown MWt: 75,700; pI: 6.20 IEF 310 SYG GLYCYL-TRNAP41250 SYNTHETASE (EC 6.1.1.14) IEF 329 GR78 78 KD GLUCOSE P06761REGULATED PROT. PREC. (GRP 78) IEF 329 NCPR NADPH-CYTOCHROME P00388 P450REDUCTASE (EC 1.6.2.4) IEF 330 GR75 MITOCHONDRIAL P48721 STRESS-70PROTEIN PRECURSOR (MORTALIN) IEF 342 Unknown MWt: 83,300; pI: 4.81 IEF347 GR78 78 KD GLUCOSE P06761 REGULATED PROT. PREC. (GRP 78) IEF 354Unknown MWt: 70,400; pI: 4.30 IEF 382 Unknown MWt: 63,900; pI: 6.35 IEF387 TO64 TURNED ON AFTER P47942 DIVISION, 64 KD PROT. (TOAD-64) IEF 387TCPG T-COMPLEX PROTEIN 1, P80318 GAMMA SUBUNIT (Mouse/Human) IEF 387COPD COATOMER DELTA P53619 SUBUNIT (DELTA-COAT PROTEIN) (Bovine/Human)IEF 425 HS7C HEAT SHOCK COGNATE P08109 71 kD PROTEIN IEF 471 UnknownMWt: 60,700; pI: 6.08 IEF 480 Unknown MWt: 62,000; pI: 5.91 IEF 483 ER60PROTEIN DISULFIDE P11598 ISOMERASE ER-60 PRECURSOR (EC 5.3.4.1) (ERP60)IEF 505 Unknown MWt: 61,500; pI: 5.41 IEF 506 TCPE T-COMPLEX PROTEIN 1,P80316 EPSILON SUBUNIT (TCP- 1-EPSILON) (Mouse) IEF 507 P60MITOCHONDRIAL P19227 MATRIX PROTEIN P1 PRECURSOR (HSP-60) IEF 561 ENOAALPHA ENOLASE (EC P04764 4.2.1.11) IEF 563 Unknown MWt: 48,000; pI: 6.20IEF 655 LAMA LAMIN A S47890 IEF 759 HSU36764 TGF-β RECEPTOR U36764INTERACTING PROTEIN 1 (HUMAN) IEF 1081 Unknown MWt: 63,800; pI: 5.43 IEF1196 Unknown MWt: 143,100; pI: 5.41; Mass spec FIGS. 20–23 IEF 1342Unknown MWt: 53,400; pI: 4.64 IEF 1356 Unknown MWt: 147,900; pI: 5.40NEPHGE G3P GLYCERALDEHYDE 3- P04797 017 PHOSPHATE DEHYDROGENASE (EC1.2.1.12) (GAPDH) NEPHGE PTB POLYPYRIMIDINE Q00438 156 TRACT-BINDINGPROTEIN (PTB) (HNRNP I) NEPHGE F261 6-PHOSPHOFRUCTO-2 P07953 169 KINASE(EC 2.7.1.105) NEPHGE PGK2 PHOSPHOGLYCERATE P16617 203 KINASE (EC2.7.2.3), NEPHGE ANX2 ANNEXIN II Q07936 269 (LIPOCORTIN II) (CALPACTIN IHEAVY CHAIN) NEPHGE G3P GLYCERALDEHYDE 3- P04797 269 PHOSPHATEDEHYDROGENASE (EC 1.2.1.12)(GAPDH) NEPHGE LEG3 GALECTIN-3 P08699 298(GALACTOSE-SPECIFIC LECTIN 3) NEPHGE Unknown MWt: 36,400; pI: 8.32 298NEPHGE ALFA FRUCTOSE- P05065 668 BISPHOSPHATE ALDOLASE (EC 4.1.2.13) A(MUSCLE) NEPHGE Unknown MWt: 148,700; pI: 8.10 670 NEPHGE Unknown MWt:132,500; pI: 8.52 672 NEPHGE Unknown MWt: 132,500; pI: 8.46 673 NEPHGEUnknown MWt: 132,500; pI: 8.42 674

TABLE 2 ISLET CELL DIABETES MEDIATING PROTEINS WHICH DECREASE UPON IL-18STIMULATION IDENTIFIED BY 2DGE Database Gel Spot No. Gene ProteinAccession IEF 015 Unknown MWt: 75,400; pI: 6.01 IEF 339 GR78 78 KDGLUCOSE P06761 REGULATED PROTEIN PRECURSOR (GRP 78) IEF 340 GR75MITOCHONDRIAL P48721 STRESS-70 PROTEIN PRECURSOR (GRP 75) IEF 344 GR7878 KD GLUCOSE P20029 REGULATED PROTEIN PRECURSOR (GRP 78) IEF 358Unknown MWt: 77,000; pI: 4.11 IEF 436 NEC2 NEUROENDOCRINE P28841CONVERTASE 2 PRECURSOR (EC 3.4.21.94) (NEC 2) IEF 441 NEC2NEUROENDOCRINE P28841 CONVERTASE 2 PRECURSOR (EC 3.4.21.94) (NEC2) IEF442 NEC2 NEUROENDOCRINE P28841 CONVERTASE 2 PRECURSOR (EC 3.4.21.94)(NEC 2) IEF 484 ER60 PROTEIN DISULFIDE P11598 ISOMERASE ER60 PRECURSOR(EC 5.3.4.1) (ERP60) IEF 510 Unknown MWt: 54,400; pI: 5.27 IEF 614 ERP5PROBABLE PROTEIN Q63081 DISULFIDE ISOMERASE P5 PRECURSOR (EC 5.3.4.1)IEF 614 ATPB ATP SYNTHASE BETA P10719 CHAIN, MITOCHONDRIAL PRECURSOR (EC3.6.1.34) IEF 665 MTA1 METASTASIS- Q62599 ASSOCIATED PROTEIN MTA-1 IEF719 Unknown MWt: 39,600; pI: 6.55 IEF 825 Unknown MWt: 35,000; pI: 5.10IEF 831 Unknown MWt: 34,600; pI: 4.76; Mass spec FIG. 41 IEF 882 UnknownMWt: 30,800; pI: 4.94 IEF 887 Unknown MWt: 30,800; pI: 4.76 IEF 895Unknown MWt: 28,900; pI: 4.29 IEF 908 ER31 ENDOPLASMIC P5255 5 RETICULUMPROTEIN ERP31 PRECURSOR (ERP29) IEF 908 PMGB PHOSPHOGLYCERATE P25113MUTASE, BRAIN FORM (EC 5.4.2.1) IEF 939 Unknown MWt: 25,900; pI: 5.09;Mass spec FIGS. 16 IEF 941 PBP PHOSPHATIDYLETHANO P31044 LAMINE-BINDINGPROTEIN (P23K) IEF 949 RNU53882 14-3-3 PROTEIN EPSILON U53882 ISOFORMIEF 949 Unknown MWt: 26,800; pI: 4.49 Mass spec FIGS. 42–43 IEF 950Unknown MWt: 25,800; pI: 4.53; Mass spec FIGS. 19 NEPHGE 001 KPY2PYRUVATE KINASE, M2 P11981 ISOZYME (EC 2.7.1.40) NEPHGE 007 Unknown MWt:65,500; pI 7.28; Mass spec FIG. 24 NEPHGE 009 Unknown MWt: 115,700; pI8.33; Mass spec FIG. 25 NEPHGE 018 DHE3 GLUTAMATE P10860 DEHYDROGENASEPRECURSOR (EC 1.4.1.3) (GDH) NEPHGE 102 TCPZ T-COMPLEX PROTEIN 1, P80317ZETA SUBUNIT NEPHGE 102 RNPKMPS PYRUVATE KINASE M M24361 NEPHGE 123 KPY2PYRUVATE KINASE, M2 P11981 ISOZYME (EC 2.7.1.40) NEPHGE 129 KPY2PYRUVATE KINASE, M2 P11981 ISOZYME NEPHGE 129 Unknown MWt: 57,600; pI:7.72 Mass spec FIGS. 44–46 NEPHGE 130 MMSA METHYLMALONATE- Q02253SEMIALDEHYDE DEHYDROGENASE PRECURSOR NEPHGE 130 RNPKMPS PYRUVATE KINASEM M24361 INTRONLESS PROCESSED PSEUDOGENE NEPHGE 171 ATPA ATP SYNTHASEALPHA P15999 CHAIN, MITOCHONDRIAL PRECURSOR (EC 3.6.1.34) NEPHGE 174DHE3 GLUTAMATE P10860 DEHYDROGENASE PRECURSOR (EC 1.4.1.3) (GDH) NEPHGE176 ATPA ATP SYNTHASE ALPHA P15999 CHAIN, MITOCHONDRIAL PRECURSOR (EC3.6.1.34) NEPHGE 181 Unknown MWt: 49,400; pI: 7.40; Mass spec FIGS.32–33 NEPHGE 182 DHE3 GLUTAMATE P10860 DEHYDROGENASE PRECURSOR (EC1.4.1.3) (GDH) NEPHGE 211 Unknown MWt: 47,900; pI: 7.28; Mass spec FIG.36 NEPHGE 227 THIL ACETYL-COA P17764 ACETYLTRANSFERASE PRECURSOR,MITOCHONDRIAL (EC 2.3.1.9) NEPHGE 231 BKCRU CREATINE KINASE, P25809UBIQUITOUS MITOCHONDRIAL PRECURSOR (EC 2.7.3.2) NEPHGE 231 BTHILACETYL-COA P17764 ACETYLTRANSFERASE PRECURSOR MITOCHONDRIAL (EC 2.3.1.9)NEPHGE 236 Unknown MWt: 43,200; pI: 7.90; Mass spec FIG. 38 NEPHGE 253Unknown MWt: 39,100; pI: 9.05; Mass spec FIGS. 39–40 NEPHGE 296 NC5RNADH-CYTOCHROME B5 P20070 REDUCTASE (EC 1.6.2.2) NEPHGE 306 KAD2ADENYLATE KINASE P29410 ISOENZYME 2, MITOCHONDRIAL (EC 2.7.4.3) NEPHUE310 Unknown MWt: 35,800; pI: 7.57; Mass spec FIG. 47 NEPHGE 326 UnknownMWt: 34,500; pI: 8.62; Mass spec FIG. 48 NEPHGE 328 Unknown MWt: 30,900;pI: 8.48 NEPHGE 334 TPIS TRIOSEPHOSPHATE P48500 ISOMERASE (EC 5.3.1.1)(TIM)

Example 8 Interleukin-1DF Induced Changes in the Protein Expression ofRat Islets: A Computerized Database

Summary

Two-dimensional (2-D) gel electrophoresis of pancreatic islet proteinscan be an important tool facilitating studies of the molecularpathogenesis of insulin-dependent diabetes mellitus. Insulin-dependentdiabetes mellitus is caused by an autoimmune destruction of the β-cellsin the islets of Langerhans. The cytokine interleukin 1β inhibitsinsulin release and is selectively cytotoxic to β-cells in isolatedpancreatic rat islets. The antigen(s) triggering the immune response aswell as the intracellular mechanisms of action of interleukin1β-mediated β-cell cytotoxicity are unknown. However, previous studieshave found an association with alterations in protein synthesis. Thus,2-D gel electrophoresis of islet proteins can lead to 1) theidentification of primary antigen(s) initiating the immune destructionof the β-cells 2) the determination of qualitative and quantitativechanges in specific islet proteins induced by cytokines and 3) thedetermination of the effects of agents modulating cytokine action.Therefore, the aim of this study was to create databases of allreproducibly detectable protein spots on 10% and 15% acrylamide 2-D gelsof neonatal rat islets (10% & 15% DB), labelled under standardizedculture conditions. 1792 spots were present in 5 of 5 gels in the 15%DB, whereas 1373 spots were present in 5 of 5 gels in the 10% DB,yielding a qualitative reproducibility between 75.2% and 91.7%. In bothDBs, the average coefficient of variation of the percentage ofintegrated optical density (CV % of % IOD) for spots present in all gelwas between 42.4% and 45.7%. When the same sample was analyzed inconsecutive sets of gels on different days (interassay analysis), theaverage CV % of % IOD was 35.5%–36.1%. When the same sample was analyzedrepeatedly in one set of gels (intraassay analysis), the average CV % of% IOD was 30.2% in the IEF gels, while the average CV % of % IOD wasunchanged (45.7%) in the NEPHGE gels. Applying the 10% DB to distinguishproteins altered in expression by IL-1β, 105 currently unidentifiedprotein spots were found to be up-/down-regulated or synthesized de novoby IL-1β. In conclusion, we present the first 10% and 15% acrylamide 2-Dgel protein databases of neonatal rat islets of Langerhans anddemonstrate its usage to identify proteins altered in expression byIL-1β.

Introduction

The cytokine interleukin 1β inhibits insulin release and is selectivelycytotoxic to β-cells in isolated pancreatic rat islets (Mandrup-Poulsen,T, Diabetologia, in press (1996)). Active protein synthesis is a crucialpart of β-cell destruction, defense and repair after insults such ascytokines. The free radical nitric oxide (NO) has been demonstrated tobe an important mediator of the deleterious effects of cytokines onislet α-cells (Southern, et al., FEBS. Lett. 276:4244 (1990); Welsh, etal., Endocrinol 129:3167–3173 (1991); Corbett, et al., J. Biol. Chem.266:21351–21354 (1991)). Thus, analogues of L-arginine, the substratefor NO production, prevent the deleterious effects of interleukin 1β(IL-1β) (Southern, et al., FEBS. Lett. 276:42–44 (1990); Welsh, et al.,Endocrinol. 129:3167–3173 (1991); Corbett, et al., J. Biol. Chem.266:21351–21354 (1991)) and mRNA for the cytokine-inducible isoform ofNO synthase (iNOS) is induced by IL-1β in β-, but not α-cells (Corbett,et al., J. Clin. Invest. 90:2384–2391 (1992)). We have recently clonediNOS from neonatal rat islets and have demonstrated the expression ofthe recombinant iNOS as a series of spots on two-dimensional (2-D) gels,most likely as phosphorylated isoforms, with the expected molecular massof 131 kDa and pI values in the range of 6.8 to 7.0 (Karlsen, et al.,Diabetes 44:753–758 (1995)).

Further, inhibitors of protein synthesis block the inhibitory effect ofIL-1β on islet function (Hughes, et al., J. Clin. Invest. 86:856–863(1990)), indicating that de novo protein synthesis is necessary for thedeleterious effect of IL-1 β. IL-1β also induces the synthesis of theheat shock proteins (HSP) HSP32 (heme oxygenase) and HSP70 (Helqvist, etal., Acta Endocrinol. (Copenh) 121:136–140 (1989); Helqvist, et al.,Diabetologia 34:150–156 (1991); Welsh, et al., Autoimmunity 9:33–40(1991)), known to play a role in protection against cellular stress andin cell repair (Kaufmann, Immunol. Today 11:129–136 (1990)). Further,IL-1β inhibits the synthesis of a number of unknown proteins withmolecular weights of 45, 50 (Hughes, et al., J. Clin. Invest. 86:856–863(1990)), 75, 85, 95 and 120 kDa (Welsh, et al., Autoimmunity 9:3340(1991)) in islets. Using 2-D gel electrophoresis, we recentlydemonstrated that IL-1β up- and downregulated 29 and 3 proteins,respectively, in neonatal rat islets.

Endocrine islet cells can play an important role in β-cell destructionand, possibly, survival. Dispersion and sorting of islet cells is apotentially harmful procedure that could influence the protein synthesispattern. The disadvantage of the chosen islet cell material is that anychange in protein expression in one cell type will appear smallerbecause it is diluted by synthesis from other cells.

Thus, the aims of this study were to determine the spot detectionreproducibility and to calculate the coefficient of variation of thepercentage of integrated optical density (CV % of % IOD) for all(³⁵S)-methionine-labelled islet protein database spots. Additionally, wewanted to investigate the contribution of the intra- and interassayvariation of the gel preparation to the total CV % of % IOD of thespots. Finally, we wanted to define the number of IL-1β-induced changesin the islet protein pattern by computer analysis.

Materials and Methods

Reagents. DMEM, RPMI 1640 and Hanks' balanced salt solution (HBSS) werepurchased from Gibco, Paisley, Scotland. RPMI 1640 was supplemented with20 mM HEPES buffer, 100,000 IU/I penicillin and 100 mg/L streptomycin.Authentic recombinant human IL-1β was provided by Novo Nordisk Ltd.(Bagsvaerd, Denmark). The specific activity was 400 U/ng (Moelvig, etal., Scand J. Immunol. 31:225–235 (1990). The following other reagentswere used: 2-mercaptoethanol, bovine serum albumin (BSA), Tris HCl, Trisbase, glycine, (Sigma, St. Louis, USA); trichloracetic acid (TCA),phosphoric acid, NaOH, glycerol, n-butanol, bromophenol blue (Merck,Darmstadt, Germany); (³⁵S)-methionine (SJ 204, specific activity: >1.000Ci/mmol, containing 0.1% 2-mercaptoethanol), Amplify® (AmershamInternational, Amersham, UK); filters (HAWP 0.25 μm pore size)(Millipore, Boston, USA); RNA'se A, DNA'se I (Worthington, Freehold,N.J., USA); urea (ultra pure) (Schwarz/Mann, Cambridge, Mass., USA);acrylamide, bisacrylamide, TEMED, ammonium persulphate (BioRad,Richmond, Calif., USA); ampholytes: pH 5–7, pH 3.5–10, pH 7–9, pH 8–9.5(Pharmacia, Uppsala, Sweden); Nonidet P-40 (BDH, Poole, UK); ampholytes:pH 5–7 and sodium dodecyl sulphate (Serva, Heidelberg, Germany); agarose(Litex, Copenhagen, Denmark); ethanol (absolute 96%) (Danish Distillers,Aalborg, Denmark); methanol (Prolabo, Brione Le Blanc, France); aceticacid (technical quality, 99% glacial) (Bie & Berntsen, Århus, Denmark)and X-ray film (Curix RP-2) (AGFA).

Islet isolation and culture. For the database and assay variationexperiments, 12 different islet isolations were performed, 10 for thedatabases, 1 for intraassay and I for interassay analysis. For thestudies involving IL-1β, 3 additional islet isolations were performed.

Islets from pancreata of 4 day old inbred Wistar Furth rats (Møllegård,Lille Skensved, Denmark) were isolated after collagenase digestion(Brunstedt, In: Lamer, J., Polh, S. L. (Eds.), Methods In DiabetesResearch Vol. 1 (Laboratory methods, Part C). Wiley & Sons, New York,pp. 254–288 (1984)). After a preculture period of 4 days in RPMI1640+10% fetal calf serum, 150 islets were incubated for 24 h in 300 μlRPMI 1640+0.5% normal human serum (NHS). In a separate series ofexperiments, 150 islets were incubated for 24 h in 300 μl RPMI 1640+0.5%NHS with or without the addition of 150 pg/ml IL-1β.

Islet labelling. After 24 h in culture, the 150 islets were harvested,washed twice in HBSS and labelled for 4 h in 200 μl methionine-freeDulbecco's modified Eagle's's medium (DMEM) with 10% NHS dialysed foramino acids, and 200 μCi (³⁵S)-methionine. To eliminate2-mercaptoethanol (³⁵S)-methionine was freeze-dried for at least 4 hbefore labelling. After labelling, islets were washed thrice in HBSS,pelleted and frozen at −80° C.

Sample preparation. The frozen islets were resuspended in 100 μl DNAseI/RNAse A solution and lysed by freeze-thawing twice. After the secondthawing they were left on ice for 30 min for the digestion of nucleicacids. The lysed sample was then freeze dried overnight. The sampleswere dissolved by shaking in 120 μl lysis buffer (8.5 M urea, 2% NonidetP-40, 5% 2-mercaptoethanol and 2% ampholytes pH range 7–9) for a minimumof 4 h.

Determination of (³⁵S)-methionine incorporation. The amount of(³⁵S)-methionine incorporation was quantitated in duplicate by adding 10μg BSA (0.2 μg/ml H₂O) as a carrier to 5 μl of a 1:10 dilution of eachsample, followed by 0.5 ml of 10% TCA. This was left to precipitate for30 min at 40° C. before being filtered through 0.25 μm filters. The HAWPfilters were dried and placed into scintillation liquid for counting.2-D gel electrophoresis. The procedure was essentially as described byO'Farrell et al., Cell 12:1133–1142 (1977) and Fey, S. J. et al., Theprotein variation in basal cells and certain basal cell related benignand malignant diseases, Faculty of Natural Science, University ofAarhus, Denmark (1984). Briefly, first dimension gels contained 4%acrylamide, 0.25% bisacrylamide and ampholytes (the actual ratiodepending upon the batch) and were 175 mm long and 1.55 mm in diameter.Equal numbers of counts (106 cpm) of each sample were applied to thegels. In case of lower amounts of radioactivity it was necessary toregulate the exposure time of the gel so that comparable total opticaldensities were obtained. The samples were analyzed on both isoelectricfocusing (IEF; pH 3.5–7) and non-equilibrium pH-gradient electrophoresis(NEPHGE; pH 6.5–10.5) gels. IEF gels were prefocused for approximately 4h at 140 μA/gel (limiting current), the sample was then applied andfocused for 18 h at 1200 V (limiting voltage). NEPHGE gels were focusedfor approximately 6.5 h using 140 μA/gel and 1200 V as the limitingparameters.

Second dimension gels, 1 mm thick, 200 mm long and 185 mm wide containedeither 15% acrylamide and 0.075% bisacrylamide or 10% acrylamide and0.05% bisacrylamide and were run overnight. After electrophoresis, thegels were fixed in 45% methanol and 7.5% acetic acid for 45 min andtreated for fluorography with Amplify® for 45 min before being dried.The gels were placed in contact with X-ray films and exposed at −70° C.for 1 to 40 days. Each gel was exposed for at least 3 time periods tocompensate for the lack of dynamic range of X-ray films.

Determination of MW and pI Molecular weights of the proteins weredetermined by comparison with standard gels (Fey, S. J. et al., Theprotein variation in basal cells and certain basal cell related benignand malignant diseases, Faculty of Natural Science, University ofAarhus, Denmark (1984)). pI for the individual proteins on the gels wasdetermined by the use of pI calibration kits. Landmark proteins wereidentified on gels by one or several of the following techniques:immunoblotting, immunoprecipitation, microsequencing or peptide mapping.

Experimental design. The study comprised three different series ofanalyses: database, intra- and interassay analysis. For each analysis,IEF and NEPHGE gels were run using 10% and 15% acrylamide in the seconddimension. This gave us four subgroups: 10% IEF; 15% IEF; 10% NEPHGE;15% NEPHGE. On 10% acrylamide gels, the approximate MW range ofdetection were between 20 and 250 kDa, while the approximate range ofdetection was between 6 and 125 kDa on 15% acrylamide gels.Consequently, proteins with a MW between 20 and 125 kDa were included inboth databases, whereas proteins with lower and higher MW wereparticular to 15% and 10% DBs, respectively. Comparison of 10% and 15%DBs revealed a lower number of detectable spots in both 10% IEF andNEPHGE subgroups (see Results). Consequently, intra- and interassayanalysis (see below) were only performed on 15% IEF and NEPHGE gels.

The databases were based on 10 different isolates analyzed in one set ofgels. After 2-D gel electrophoresis, 5 gels with the best technicalquality and with comparable optical densities were chosen for computeranalysis. Before computer analysis, one gel in each subgroup wasarbitrarily selected to be the “master gel” used for comparison with theother 4 database gels, the 5 intraassay gels and the 5 interassay gels.The database “master gel”was used as a master for intra- and interassayanalysis to ensure that a given spot had the same match number in thethree series of analyses. Data from the “master gel” are only includedin the database analysis. The “master gel” was from the same isolate inall 4 subgroups, whereas the identity of the isolates producing the 4other database gels varied slightly from subgroup to subgroup (Table 3).

For intraassay analysis, 10 gels of the same sample were analyzed in oneset of gels. After 2-D gel electrophoresis, 5 gels with the besttechnical quality and with comparable optical densities were chosen forcomputer analysis (Table 3).

For interassay analysis, the same sample was analyzed in 10 consecutivesets of gels on different days. After 2-D gel electrophoresis, 5 gelswith the best technical quality and with comparable optical densitieswere chosen for computer analysis (Table 3).

For identification of proteins altered in expression by IL-1β, 10% IEFand NEPHGE gels of IL-1β exposed islets, previously analyzed visually(Andersen, et al., Diabetes 44:400–407 (1995)), were matched to the 10%IEF and NEPHGE DBs.

Computer analysis of fluorographs. Computer analysis was performed usingthe BioImage® program (version 4.6 M) on a Sunsparc workstation. First,the fluorographs were scanned and spots were identified and quantitatedby the BioImage® program BioImage, Ann Arbor, Mass., USA. Next, eachnon-master gel was compared to the “master gel” and manually edited toensure identification and quantitation of spots not found initially bythe computer program. This comparison was performed by the same observer(H.U.A.) using the BioImage® program. Following this, the gels werematched by the BioImage® program and the accuracy of the match inspectedand corrected by the same observer. Finally, data were extracted forcalculations in the Quattro Pro® spreadsheet (Borland version 4.0).

To avoid the presence of duplicate spots in the IEF and NEPHGEsubgroups, overlapping spots in either the basic part of IEF gels or inthe acidic part of NEPHGE gels were omitted from analysis in thedatabases and the assay analyses.

Statistical analysis. Two different analyses were applied to distinguishthe proteins altered in expression by IL-1β. In the first analysis, analteration was considered significant if the average % IOD of a spot inIL-1β-exposed gels was higher or lower than the average % IOD±2 SD ofthe same spot in the DB. In the second comparison between the twogroups, Student's t test was applied and P<0.01 was chosen as level ofsignificance.

Qualitative reproducibility of the neonatal rat islet protein databasesand assay analyses. 1293 to 1411 (IEF) and 605 to 764 (NEPHGE) spotswere found in the individual gels used to construct the 15% DB, whereas1101 to 1200 (IEF) and 462 to 577 (NEPHGE) spots were found in the gelsused for the 10% DB (Tables 4 and 5). “Master gels” were made for the10% IEF and NEPHGE DB. In total, 1792 spots were present in 5 of 5 gelsin the 15% DB, whereas 1373 spots were present in 5 of 5 gels in the 10%DB, yielding a qualitative reproducibility (the average of thepercentage of spots found in 5 of 5 gels) in the subgroups between 75.2%(NEPHGE 10%) and 91.7% (IEF 15%) (Tables 4 and 5) (For each spot presentin 5 of 5 gels, the databases consist of spot match number, % IOD forthe 5 individual spots, average % IOD, standard deviation of % IOD, CV %of % IOD, MW and pI.).

As demonstrated in Tables 4 and 5, the total number of spots in theindividual gels as well as the number and percentage of spots present in5 of 5 gels were fewer in the 10% DB than in the 15% DB. However, if thedatabases were extended to include spots present in at least 3 of 5gels, no differences in the percentage of spots present were foundbetween the two databases (IEF: 15%: 98.8±1.2; 10%: 97.4±1.5; NEPHGE:15%: 94.8±5.7; 10%: 94.1±3.5, Tables 4 and 5). In both databases, thepercentage of spots present in 5, 4, 3 or 2 of 5 gels were lower inNEPHGE gels than in IEF gels (Tables 4 and 5). The spatial location ofthe spots present in less than 5 gels was investigated, demonstratingthat the spots were not grouped in specific areas of the gels dependingon whether the spot was present in 1, 2, 3 or 4 gels.

Intra- and interassay analyses were only performed on 15% gels, sincethe number of detectable spots was higher in this database. In bothanalyses, the number of spots in the individual gels as well as thenumber and percentage of spots present in 5 of 5 gels were slightly(9%–19%) reduced compared to the 15% DB (compare Tables 4 and 6).

Quantitative reproducibility of the neonatal rat islet protein databasesand assay analyses. The quantitative reproducibility was defined as theaverage of the CV % of % IOD for each spot present in 5 of 5 gels. Forthe databases, the average CV % was at a comparable level (42.4%–45.7%)in both 10% and 15% IEF and NEPHGE subgroups of gels (Table 7). For allDB subgroups the CV % ranged between 3.0% and 167.9% (Table 7). Forinterassay analyses, the average CV % were 35.5%–36.1% for both IEF andNEPHGE gels, whereas the average CV % was 30.2% for the intraassayanalysis of IEF gels and 45.7% for NEPHGE gels.

Subsequently, the database spots present in all gels were ranked inincreasing order of CV % of % IOD, resulting in similar sigmoid-shapedcurves for spots in all four database subgroups. Thus, 30% of the spotshad a CV % that was lower than 29.7%–32.5%, 50% of the spots had a CV %that was lower than 37.8%–42.8%, 90% of the spots had a CV % that waslower than 68.4%–80.6%. The slopes of the curves indicate that the5%–10% spots with the highest CV % contribute significantly to theaverage CV % of % IOD. This is supported by the fact that the medianvalues of the database subgroups are 2.3% to 5.5% lower than the meanvalues of the subgroups (Table 7).

Regression analyses between the average % IOD and CV % of % IOD for eachspot in the database subgroups. In the NEPHGE 10% and 15% DB subgroups,2 and 6, respectively, of the 10 spots with the highest average IOD %were found in the percentile with the lowest CV % (see above). Althoughnone of the 10 spots with the highest average IOD % were found in thispercentile in the IEF DB subgroups, regression analyses were performedto investigate whether a correlation existed between spot average % IODand CV %. Regression analyses demonstrated that a significant negativecorrelation existed between these two parameters (range: p=O (IEF10%)-p=0.00288 (IEF 15%)). However, since the R²-values were very lowfor all subgroups (range: R²=0.0072 (IEF 15%) R ²=0.0317 (IEF 10%)), themajority of the variability-of CV % is not explained by variation inaverage % IOD.

Application of the 10% IEF and NEPHGE DB to distinguish proteins alteredin expression by IL-1β. In a recent paper, we demonstrated that IL-1βBup and downregulated 29 and 4 proteins, respectively in 2-D gels ofneonatal rat islet proteins (Andersen, et al., Diabetes 44:400–407(1991)). 10% gels were prepared from (³⁵S)-methionine labelled WistarFurth neonatal rat islets cultured under similar conditions as thepresent study. Consequently, the rat islet 10% IEF and NEPHGE DB wasused for comparison with the computer analyzed gels of IL-1β-exposedislets, analyzed visually in the previous paper (Andersen, et al.,Diabetes 44:400–407 (1991)). Using ±2 SD of IOD % of each DB spot as acutoff level (comparable to the criterion for significant up- or downregulation in the visual analysis), comparison with the 10% DB confirmed32 of these alterations and as expected identified several new proteinchanges. Thus, a total of 183 spots were upregulated, 113 downregulatedand 34 synthesized de novo by IL-1β (results not shown). When usingp<0.01 as a cutoff level in a Student's t test, the final analysisshowed that 52 spots were upregulated, 47 downregulated- and 6synthesized de novo by IL-1β, 13 of these included in the 33 spotsselected by visual analysis.

Discussion:

In this study, we present a 10% and 15% acrylamide 2-D gel protein DB ofneonatal rat islets of Langerhans, comprising the first proteindatabases of islets or insulin secreting cells in any species. 1792spots were present in 5 of 5 gels in the 15% DB, whereas 1373 spots werepresent in 5 of 5 gels in the 10% DB, yielding a qualitativereproducibility between 75.2% and 91.7%. In both databases, the averageCV % of % IOD was between 42.4% and 45.7%. Applying the 10% DB todistinguish proteins altered in expression by IL-1β, 105 currentlyunidentified protein spots were found to be up-/down-regulated orsynthesized de novo by IL-1β.

Characteristics of neonatal Wistar Furth rat islets. To reducevariability, the inbred Wistar Furth strain of rats was chosen as anislet donor for our databases. This strain is the inbred variant of theoutbred Wistar routinely used for islet experiments in our lab(Andersen, et al., Diabetes 43:770–777 (1994)). We have previouslydetermined that the function of Wistar Furth neonatal rat isletscultured with or without IL-1β is comparable to that of Wistar neonatalrat islets (Andersen, et al. Diabetes 44:400–407 (1995); Andersen, etal., Acta Endocrinol. 120:92–98 (1989)) and have determined the effectsof IL-1β on the 2-D gel protein pattern of Wistar Furth islets(Andersen, et al. Diabetes 44:400–407 (1995)). Since the presentdatabases are based on neonatal, and not adult rat islets, we can notexclude that the protein pattern of adult islets will be different.However, adult and neonatal islets from outbred Wistar rats are equallysensitive to the deleterious effect of IL-1. (Mandrup-Poulsen, et al.,Diabetes 36:641–647 (1987)).

Each litter of newborn rats used for islet isolation typically consistsof 8–12 pups with a varying frequency of males and females. Sincecomparison of Coomassie Blue-stained gels of liver proteins from maleand female outbred Wistar rats revealed quantitative differences in 7 of250 analyzed spots and since six proteins were found exclusively inmales and one protein exclusively in females (Steiner, et al.,Electrophoresis 16:1969–1976 (1995)), it is likely that some of theproteins in our database are gender-specific or gender-regulated.Consequently, it is possible that the high variation of some of thespots in our databases could be reduced if we had chosen to constructseparate databases of islets from male and female rats. However, thegels of liver proteins were performed on non-cultured cells which couldmean that the sex-determined protein variability could be induced bycirculating sex steroids and not an inherent trait of the liver cellsper se. Circulating hormones are not likely to interfere in our proteinpattern since I) we preculture our islets for 4 days before experimentsand II) no differences in serum concentrations of sex steroids are foundbefore puberty. Further, we have previously demonstrated that isletsfrom male and female outbred Wistar rats are equally sensitive to thedeleterious effect of IL-1 (Steiner, et al., Electrophoresis16:1969–1976 (1995)).

Detection of islet proteins. Not all spots detected in our databaseswill represent different protein entities, since some spots canrepresent modifications (e.g. acetylation, methylation, phosphorylationor carbamylation) of other proteins. However, the detected number ofspots is an underestimation of the total number of islet proteins, sincethe protein database does not include proteins below the limit ofsensitivity, proteins not containing methionine, proteins with amolecular weight below 6 kDa or above 250 kDa or proteins with a pHbelow 3.5 or above 10.5. Further, about 40% of the spots with IODs abovelimits of detection have previously been estimated to be missed becausethey are obscured by other spots (Garrels, J. Biol. Chem. 264:5269–5282(1989)). Finally, the 4h labelling period favours the labelling ofproteins with high synthesis rates, whereas longer labelling periodscould be required to produce databases where all proteins are insteady-state.

Qualitative reproducibility. Previous reports of the qualitativereproducibility of 2-D gel protein databases are few and the resultsvariable: In a mouse liver protein database of Coomassie Blue-stained2-D gels, 826 spots were present in the master image and on the average500 spots were matched in 85% of the other mouse liver patterns(Giometti, et al., Electrophoresis 13:970–991 (1992)). In proteindatabases of (³⁵S)-methionine labelled mouse embryos over 80% of spotsin each of the four gel images were automatically matched to thestandard image (Shi, et al., Molec. Reprod Develop. 37:34–47 (1994)). Inour study, 1792 spots (75.2%–91.7%) were present in 5 of 5 gels in the15% DB, whereas the average percentage of spots present in 5 of 5 gelswas 5–10% lower in the 10% DB. This is presumably due to the fact thatfewer proteins exist in the high molecular weight region only analyzableon the 10% gels than in the low molecular weight region only analyzableon the 15% gels. In all groups analyzed, the qualitative reproducibilityof NEPHGE gels was lower than IEF gels. Since NEPHGE gels, contrary toEF gels, are non-equilibrium gels the risk that identical spots have aslightly different horizontal location is increased. However, our manualediting have ascertained that this problem has been eliminated as muchas possible.

Quantitative reproducibility. Regarding the quantitativereproducibility, comparisons with other studies are difficult, since themethods-used for spot identification are not identical. Further, thespots included in calculations of CV % of % IOD in most of thepreviously published databases are selected from the total number ofmatched spots according to varying criteria. In ten CoomassieBlue-stained gels of male and female Wistar rat liver proteins, 250 ofmore than 1,000 spots present in the “master gel” were selectedaccording to good shape, size and resolution and the presence and goodquality in previous experiments (Steiner, et al., Electrophoresis16:1969–1976 (1995)). Using these criteria, one third of the spots had aCV % below 20%, more than half had a CV % below 30% and three quartershad a CV % below 40% (Steiner, et al., Electrophoresis 16:1969–1976(1995)). In (³⁵S)-methionine labelled protein databases consisting of 5gels of compacted eight-cell (CEC) mouse embryos and 4 gels ofblastocyst-stage (BS) mouse embryos, 1,674 and 1,653 spots,respectively, were matched in all gels (Shi, et al., Molec. Reprod.Develop. 37:3447 (1994)). Calculated on the basis of all matched spots,the percentage error (defined as SEM×100/average) of 74% (CEC) or 79%(BS) of these spots was below 50%, and 45% (CEC) or 47% (BS) of thespots had a percentage error below 30% (Shi, et al., Molec. Reprod.Develop. 37:3447 (1994)). For comparison, conversion of SD's to SEM's(SEM=SD/√n) would give an average CV % of 20.3% in the islet IEF 15% DB,and 97.7% and 83.2% of the spots would have a percentage error below 50%and 30%, respectively.

Although the quantitative reproducibility of our study is comparable toor even better than the study in mouse embryos (Shi, et al., Molec.Reprod Develop. 37:34–47 (1994)), the average CV % of % IOD in ourdatabases are still relatively high. As previously mentioned, theheterogeneous cell population of islets and the different male/femaleratio of the islet isolations could contribute to gel variability.Although we have attempted to use gels with comparable total opticaldensities (the largest difference within each subgroup was by a factorof 3.5 (gel DB10 vs. gel DB3, IEF 15% DB, Table 3)), the non-linearsaturation of X-ray film will contribute to the size of the CV % for alldatabase spots. The application of phosphoimaging, a technique notavailable in our laboratory when this study was initiated, would reducethe contribution of this phenomenon to the magnitude of the CV %.Finally, electronic noise and differences in spot boundary definition inthe computer analysis can contribute to the magnitude of the CV %.Contrary to some other gel analysis programs, the BioImage® program usesthe local, and not the total background for boundary definition,reducing the contribution of the latter factor to the CV %.

Studies of replicate gels. In a study of 10 replicate gels of(³⁵S)-methionine labeled REF 52 cells, Garrels selected 1109 of the mostprominent spots out of a total of approximately 2,000 spots and found anaverage CV % of 26.5%, with a range between <5% and >100% and a modalvalue between 10% and 15% (Garrels, J. Biol. Chem. 264:5269–5282(1989)). It is unclear whether the samples were analyzed in consecutiveor the same set of gels. When grouping the spots according to spotquality (fitting to Gaussian shapes, overlapping of neighboring spots)and omitting spots with low density in all gels, the 19.1% spots of thehighest quality had an average CV % of 13.0% (Garrels, J. Biol. Chem.264:5269–5282 (1989)). As expected, the average CV % of % IOD wasreduced when the 15% IEF and NEPHGE interassay analyses were compared tothe 15% IEF DB, the reduction being by approximately 9%. Since theday-to-day variation of gel preparation was eliminated in the intraassayanalyses, the average CV % was expected to decrease even more. In the15% IEF subgroup, CV % was decreased by =15% compared to the database,whereas no decrease was found in the 15% NEPHGE subgroup. The reason forthe high average CV % in the 15% NEPHGE intraassay subgroup, which alsohas the lowest qualitative reproducibility of all subgroups (Tables4–6), is unknown. As the database gels were also analyzed in one set ofgels, the fraction of the CV % that is attributable to biologicalvariation should be given by the difference in CV % between database andintraassay analysis for a given spot. Thus, if the result of the 15%NEPHGE intraassay analysis is disregarded, approximately one third ofthe average CV % of % IOD is due to biological variation.

Effects of IL-1β on islet protein expression. IL-1β altered theexpression of 105 so far unidentified proteins. IL-1 mechanism of actionon islet cells is not fully clarified, but three distinct groups ofproteins might play important roles: proteins participating insignal-transduction and proteins encoded by so-called early response andlate response genes (Eizirik, et al., Diabetologia 39:875–890 (1996)).IL-1β-induced signal transduction in target cells is thought to involvefour major signalling pathways: nuclear factor-Kb, the stress-activatedprotein kinases (SAPK/JNK), protein kinase C and tyrosine kinase(Mandrup-Poulsen, T., Diabetologia 39:1005–1029 (1996); Eizirik, et al.,Diabetologia 39:875–890 (1996)). The three pathways lead to a rapid andtransient induction of the early response genes of which c-fos, c-junand interferon response factor-1 have been implicated in cytokine actionon islet cells. The early response genes activate specific genes withpossible deleterious (iNOS, cycloxygenase-2 and lipoxygenase) andprotective (HSP72, haem oxygenase, Mn superoxide dismustase) action onislets (Mandrup-Poulsen, T., Diabetologia, 39:1005–1029 (1996); Eizirik,et al., Diabetologia 39:875–890 (1996)). Thus, the information aboutIL-1β mechanism of action in islet cells is still limited and theidentification of the 105 proteins altered in expression by IL-1β mightlead to new knowledge about signal transduction and proteins withprotective and deleterious actions.

CONCLUSION

We have established a protein database of neonatal rat islets ofLangerhans with a high qualitative reproducibility and a quantitativereproducibility that improves on previously published databases on othercells and tissues. Further, we have determined intra- and interassayvariations of the neonatal rat islet protein database. The database hasfurther been applied to identify proteins altered in expression byIL-1β, which might have important roles in an IL-1β mechanism of action.Since IL-1β is cytotoxic to the insulin producing rat B cells,identification of these proteins, currently being performed by massspectrometry and microsequencing, is expected to result in significantknowledge about the pathogenesis of insulin dependent diabetes mellitus.

TABLE 3 Correction factors between the total optical densities of masterand non-master gels in DB, intra- and interassay analyses of 2-D gels ofneonatal rat islet proteins. 15% gels IEF DB Interassay Intraassay gelDB10 (master): 1 gel IE3: 1 gel IA2: 1 gel DB3: 0.293 gel IE4: 1.096 gelIA3: 0.620 gel DB6: 0.303 gel IE8: 1.129 gel IA4: 0.738 gel DB8: 0.840gel IE9: 0.784 gel IA6: 1.014 gel DB9: 0.284 gel IE10: 0.804 gel IA10:0.747 NEPHGE DB Interassay Intraassay gel DB10 (master): 1 gel IE3: 1gel IA1: 1 gel DB3 0.542 gel IE4: 1.901 gel IA2 1.599 gel DB6: 1.067 gelIE8: 1.761 gel IA3: 0.841 gel DB8: 0.986 gel IE9: 1.408 gel IA4: 0.908gel DB9: 0.831 gel IE10: 1.599 gel IA5: 1.135 10% gels IEF NEPHGE DB DBgel DB10 (master): 1 gel DB10 (master): 1 gel DB1: 0.947 gel DB1: 1.660gel DB4: 0.358 gel DB7: 3.215 gel DB6: 1.167 gel DB8: 2.959 gel DB8:1.145 gel DB9: 1.197The databases were based on 10 different isolates analyzed in one set ofgels, while interassay analysis consisted of 10 gels of the same sampleanalyzed in one set of gels and interassay analysis was based on theanalysis of the same sample run in 10 consecutive sets of gels ondifferent days. Before computer analysis, one gel in each databasesubgroup was arbitrarily selected to be the “master gel” used forcomparison with the other 4 database gels, the 5 intraassay gels and the5 interassay gels. The numbers (1–10) of the isolates/replicates chosenare indicated in the Table. The correction factors between the totaloptical densities of the master and non-master gels were calculated inthe BioImage® program following analysis. Gels with a correction factor<1 have a higher total optical density than the “master gel”, e.g., inthe 15% IEF DB, the total optical density of gel DB10=0.293×gel DB3. Forthe intra- and interassay analyses, correction factors were calculatedbetween an arbitrarily selected gel and the 4 other gels. Comparisoncannot be made between subgroups because gels with a correction factorof 1 not necessarily have the same intensity.

TABLE 4 Reproducibility of spot detection in 15% IEF and NEPHGE 2-DGE DBof neonatal rat islet proteins. spots in spots in spots in spots intotal no. 5 of 5 gels 4–5 of 5 gels 3–5 of 5 gels 2–5 of 5 gels of spotsno. % no. % no. % no. % IEF gel DB3 1325 1235 93.2 1299 98.0 1320 99.61325 100 gel DB6 1352 1235 91.3 1322 97.8 1346 99.6 1352 100 gel DB81293 1235 95.5 1276 98.7 1287 99.5 1292 99.9 gel DB9 1355 1235 91.1 131997.3 1339 98.8 1355 100 gel DB10 1411 1235 87.5 1327 94.0 1365 96.7 139899.1 avg ± SD 91.7 ± 3.0 97.2 ± 1.8 98.8 ± 1.2 99.8 ± 0.4 NEPHGE gel DB3663 557 84.0 604 91.1 633 95.5 658 99.2 gel DB6 605 557 92.1 584 96.5598 98.8 604 99.8 gel DB8 629 557 88.6 597 94.9 614 97.6 623 99.0 gelDB9 634 557 87.9 601 94.8 617 97.3 630 99.4 gel DB10 764 557 72.9 61079.8 648 84.4 701 91.8 avg ± SD 85.1 ± 7.4 91.4 ± 6.8 94.8 ± 5.7 97.8 ±3.4Construction of the 2-D gel database: neonatal rat islets from 5different isolates were cultured for 24 h in RPMI 1640+0.5% HS, washedtwice and labelled for 4h with (³⁵S)methionine. Following 2-DGE (seeMaterials and Methods) in one set of gels, the fluorographs were scannedand spots were identified and quantitated by the BioImage® program. Eachgel was compared and matched to the arbitrarily selected “master gel”(gel DB 10). For each gel, the table indicates the number and percentageof spots present in (from left to right) all gels, at least 4 of 5 gels,at least 3 of 5 gels and at least 2 of 5 gels.

TABLE 5 Reproducibility of spot detection in 10% IEF and NEPHGE 2-DGE DBof neonatal rat islet proteins. spots in spots in spots in spots intotal no. 5 of 5 gels 4–5 of 5 gels 3–5 of 5 gels 2–5 of 5 gels of spotsno. % no. % no. % no. % IEF gel DB1 1101 995 90.4 1060 96.3 1070 97.21072 97.4 gel DB4 1200 995 82.9 1094 91.2 1143 95.3 1163 96.9 gel DB61120 995 88.8 1075 96.0 1106 98.8 1108 98.9 gel DB8 1119 995 88.9 108496.9 1106 98.8 1113 99.5 gel DB10 1198 995 83.1 1106 92.3 1162 97.0 119399.6 avg ± SD 86.8 ± 3.5 94.5 ± 2.6 97.4 ± 1.5 98.5 ± 1.2 NEPHGE gel DB1516 378 73.3 438 84.9 475 92.1 489 94.8 gel DB7 462 378 81.8 424 91.8442 95.7 445 96.3 gel DB8 480 378 78.8 440 91.7 468 97.5 472 98.3 gelDB9 492 378 76.8 441 89.6 474 96.3 482 98.0 gel DB10 577 378 65.5 45578.9 513 88.9 539 93.4 avg ± SD 75.2 ± 6.3 87.4 ± 5.5 94.1 ± 3.5 96.2 ±2.1Construction of the 2-D gel database: neonatal rat islets from 5different isolates were cultured for 24 h in RPMI 1640+0.5% HS, washedtwice and labelled for 4h with (³⁵S)-methionine. Following 2-DGE (seeMaterials and Methods) in one set of gels, the fluorographs were scannedand spots were identified and quantitated by the BioImage® program. Eachgel was compared and matched to the arbitrarily selected “master gel”(gel DB 10). For each gel, the table indicates the number and percentageof spots present in (from left to right) all gels, at least 4 of 5 gels,at least 3 of 5 gels and at least 2 of 5 gels.

TABLE 6 Reproducibility of spot detection in replicate 15% IEF andNEPHGE 2-D gels of neonatal rat islet proteins. spots in spots inIntraassay total no. 5 of 5 gels Interassay total no. 5 of 5 gelsanalysis of spots no. % analysis of spots no. % IEF 1289 1085 84.2 IEFgel IA2 1289 1085 84.2 gel IE3 1319 1082 82.0 gel IA3 1337 1085 81.2 gelIE4 1348 1082 80.3 gel IA4 1289 1085 84.2 gel IE8 1333 1082 81.2 gel IA61303 1085 83.3 gel IE9 1342 1082 83.2 gel IA10 1326 1085 81.8 gel IE101300 1082 80.6 avg ± SD 82.9 ± 1.4 avg ± SD 81.5 ± 1.2 NEPHGE gel IA1526 345 65.6 gel IE3 574 421 75.0 gel IA2 542 345 63.7 gel IE4 589 42173.3 gel IA3 538 345 64.1 gel IE8 566 421 71.5 gel IA4 565 345 61.1 gelIE9 590 421 74.4 gel IA5 450 345 76.7 gel IE10 561 421 71.4 avg ± SD66.2 ± 6.1 avg ± SD 73.1 ± 1.6For intraassay analysis, 5 independent gels of the same islet celllysate were analyzed in one set of gels. For interassay analysis, 5independent gels of the same islet cell lysate were analyzed inconsecutive sets of gels on different days. Different islet isolateswere used for database, intra- and interassay analysis. When analyzed inthe BioImage® program, the fluorographs were compared and matched to the15% IEF of the NEPHGE “master gel” of Table 2.

TABLE 7 Average coefficients of variance of % integrated optical densityof spots detectable in 5 of 5 gels in databases and replicate 2-D gelsof neonatal rat islet proteins. Analysis Average CV % Median (Range) CV% IEF 15% DB 45.4 ± 25.0 39.9 (5.0–165.3) IEF 15% Interassay 36.1 ± 19.832.6 (2.7–190.6) IEF 15% Intraassay 30.2 ± 17.1 27.3 (0.0–130.4) NEPHGE15% DB 44.3 ± 22.5 42.0 (3.9–155.1) NEPHGE 15% Interassay 35.5 ± 19.733.1 (2.2–118.5) NEPHGE 15% Intraassay 45.7 ± 22.8 43.9 (4.3–130.9) IEF10% DB 45.7 ± 21.3 42.7 (3.0–133.4) NEPHGE 10% DB 42.4 ± 22.4 37.7(7.3–167.9)The average coefficient of variance (CV %) was calculated from the CV %of % IOD of all spots present in 5 of 5 gels in each subgroup ofanalysis. Results are presented as means±SD (left column) and as medians(ranges). The number of spots in 5 of 5 gels in each subgroup is shownin Tables 2–4. For details of design databases and replicate analyses,please see Materials and Methods.

TABLE 8 Proteins Present in Unaffected or Normal Rat Islet Cells IEF 10%gels NEPHGE 10% gels match % IOD match % IOD no. ratio mw pI no. ratiomw pI 11 2.34 118,652 6.64 7 0.38 65,522 7.28 15 0.29 75,443 6.01 175.09 39,973 8.29 25 1.79 54,244 5.53 102 0.45 63,560 7.26 28 2.65 65,7985.06 129 0.32 57,609 7.72 83 2.56 164,145 6.34 130 0.45 55,734 8.07 854.21 164,145 6.28 156 3.22 55,550 8.71 115 2.56 175,154 5.23 169 2.1655,642 8.23 145 8.51 133,052 6.29 171 0.21 52,860 8.20 186 22.00 152,0775.01 174 0.43 53,830 7.92 187 6.19 152,077 4.95 176 0.21 52,598 7.96 1891.88 135,100 4.96 181 0.20 49,422 7.40 194 3.31 139,291 4.65 182 0.5554,098 7.61 201 2.48 143,611 4.10 203 1.51 44,362 8.19 210 3.19 114,5196.40 211 0.28 47,925 7.28 225 4.06 83,281 5.89 227 0.29 43,939 8.43 2653.31 120,019 4.99 231 0.41 44,362 8.34 267 2.12 92,500 4.78 236 0.1643,162 7.90 276 2.52 107,498 4.34 253 0.25 39,106 9.05 279 6.78 121,4014.20 269 9.04 39,863 8.01 289 1.95 73,880 6.44 296 2.96 36,169 8.29 3062.65 75,706 6.20 298 0.07 36,382 8.32 310 1.88 69,383 5.70 306 0.0935,666 8.14 329 2.23 72,856 5.27 310 0.27 35,827 7.57 330 2.74 68,8095.35 326 0.17 34,521 8.62 342 2.80 83,281 4.81 328 0.03 30,920 8.48 3546.23 70,358 4.30 334 0.11 30,920 8.17 358 0.19 76,975 4.11 382 2.5563,920 6.35 387 2.41 64,342 6.14 425 2.09 67,198 5.18 436 0.38 66,4074.76 441 0.22 66,758 4.62 442 0.16 66,934 4.53 471 2.81 60,722 6.08 4831.72 61,204 5.71 484 0.27 59,247 5.88 505 2.30 61,526 5.41 506 2.3460,007 5.42 507 2.72 59,928 5.33 510 0.26 54,485 5.27 561 2.52 49,3126.00 563 3.04 48,018 6.20 655 1.85 41,355 6.15 665 0.33 42,243 5.82 7190.69 39,558 6.55 759 2.40 37,116 5.34 825 0.13 35,027 5.10 831 0.5334,623 4.76 882 0.10 30,920 4.94 887 0.24 30,837 4.76 895 0.69 28,8934.29 908 0.27 25,753 6.28 939 0.37 25,851 5.09 941 0.08 22,704 5.15 9490.21 26,803 4.49 1,081 2.68 63,836 5.43 1,342 3.74 53,379 4.64The match numbers of the table correspond to the match numbers of the 10% IEF and NEPHGE DBs. The spot numbers given in a previous paper (i 2)to spots aftered in expression by IL-1 B are indicated in parenthesis. *indicates that the spot was not previously found to be aftered inexpression by IL-1 B alone but by other experimental conditions (1 2).For I EF and N EPHG E gels, the analysis was based on 5 DB gels and 3IL-1 B gels and P<0.01 was chosen as level of signfficance. The % IODratio expresses the average % IOD of IL-1 B gels/average % IOD of DBgels. Thus, a ratio above 1 indicates that the spot is upregulated. DNindicates that the spot is synthesized de novo by IL-1 B.

TABLE 9 Affected Proteins up-/down-regulated or synthesized de novo byIL-1B Treatment or Expected to be up-/down-regulated or synthesized denovo in Islet Cells when Immunologically Affected in IDDM. IEF 10% gelsNEPHGE 10% gels match % IOD match % IOD no. ratio mw pI no. ratio mw pI11 2.34 118,652 6.64 1 0.17 58,801 8.27 15 0.29 75,443 6.01 7 0.3865,522 7.28 25 1.79 54,244 5.53 17 5.09 39,973 8.29 28 2.65 65,798 5.06102 0.45 63,560 7.26 83 2.56 164,145 6.34 129 0.32 57,609 7.72 85 4.21164,145 6.28 130 0.45 55,734 8.07 115 2.56 175,154 5.23 156 3.22 55,5508.71 145 8.51 133,052 6.29 169 2.16 55,642 8.23 186 22.00 152,077 5.01171 0.21 52,860 8.20 187 6.19 152,077 4.95 174 0.43 53,830 7.92 189 1.88135,100 4.96 176 0.21 52,598 7.96 194 3.31 139,291 4.65 181 0.20 49,4227.40 201 2.48 143,611 4.10 182 0.55 54,098 7.61 210 3.19 114,519 6.40203 1.51 44,362 8.19 225 4.06 83,281 5.89 211 0.28 47,925 7.28 265 3.31120,019 4.99 227 0.29 43,939 8.43 267 2.12 92,500 4.78 231 0.41 44,3628.34 276 2.52 107,498 4.34 236 0.16 43,162 7.90 279 6.78 121,401 4.20253 0.25 39,106 9.05 289 1.95 73,880 6.44 269 9.04 39,863 8.01 306 2.6575,706 6.20 296 2.96 36,169 8.29 310 1.88 69,383 5.70 298 0.07 36,3828.32 329 2.23 72,856 5.27 306 0.09 35,666 8.14 330 2.74 68,809 5.35 3100.27 35,827 7.57 342 2.80 83,281 4.81 326 0.17 34,521 8.62 354 6.2370,358 4.30 328 0.03 30,920 8.48 358 0.19 76,975 4.11 334 0.11 30,9208.17 382 2.55 63,920 6.35 668 DN 42,809 8.46 387 2.41 64,342 6.14 4252.09 67,198 5.18 436 0.38 66,407 4.76 441 0.22 66,758 4.62 442 0.1666,934 4.53 471 2.81 60,722 6.08 483 1.72 61,204 5.71 484 0.27 59,2475.88 505 2.30 61,526 5.41 506 2.34 60,007 5.42 507 2.72 59,928 5.33 5100.26 54,485 5.27 561 2.52 49,312 6.00 563 3.04 48,018 6.20 655 1.8541,355 6.15 665 0.33 42,243 5.82 719 0.69 39,558 6.55 759 2.40 37,1165.34 825 0.13 35,027 5.10 831 0.53 34,623 4.76 882 0.10 30,920 4.94 8870.24 30,837 4.76 895 0.69 28,893 4.29 908 0.27 25,753 6.28 939 0.3725,851 5.09 941 0.08 22,704 5.15 949 0.21 26,803 4.49 1,081 2.68 63,8365.43 1,342 3.74 53,379 4.64 1,356 DN 147,898 5.40The match numbers of the table correspond to the match numbers of the 10% IEF and NEPHGE DBs. The spot numbers given in a previous paper (i 2)to spots aftered in expression by IL-1 B are indicated in parenthesis. *indicates that the spot was not previously found to be aftered inexpression by IL-1 B alone but by other experimental conditions (1 2).For I EF and N EPHG E gels, the analysis was based on 5 DB gels and 3IL-1 B gels and P<0.01 was chosen as level of signfficance. The % IODratio expresses the average % IOD of IL-1 B gels/average % IOD of DBgels. Thus, a ratio above I indicates that the spot is upregulated. DNindicates that the spot is synthesized de novo by IL-1 B.

TABLE 10 Marker Proteins In Islet Cells IEF 10% gels NEPHGE 10% gelsmatch % IOD match % IOD no. ratio mw pI no. ratio mw pI 11 2.34 118,6526.64 7 0.38 65,522 7.28 15 0.29 75,443 6.01 17 5.09 39,973 8.29 25 1.7954,244 5.53 102 0.45 63,560 7.26 28 2.65 65,798 5.06 129 0.32 57,6097.72 83 2.56 164,145 6.34 130 0.45 55,734 8.07 85 4.21 164,145 6.28 1563.22 55,550 8.71 115 2.56 175,154 5.23 169 2.16 55,642 8.23 145 8.51133,052 6.29 171 0.21 52,860 8.20 186 22.00 152,077 5.01 174 0.43 53,8307.92 187 6.19 152,077 4.95 176 0.21 52,598 7.96 189 1.88 135,100 4.96181 0.20 49,422 7.40 194 3.31 139,291 4.65 182 0.55 54,098 7.61 201 2.48143,611 4.10 203 1.51 44,362 8.19 210 3.19 114,519 6.40 211 0.28 47,9257.28 225 4.06 83,281 5.89 227 0.29 43,939 8.43 265 3.31 120,019 4.99 2310.41 44,362 8.34 267 2.12 92,500 4.78 236 0.16 43,162 7.90 276 2.52107,498 4.34 253 0.25 39,106 9.05 279 6.78 121,401 4.20 269 9.04 39,8638.01 289 1.95 73,880 6.44 296 2.96 36,169 8.29 306 2.65 75,706 6.20 2980.07 36,382 8.32 310 1.88 69,383 5.70 306 0.09 35,666 8.14 329 2.2372,856 5.27 310 0.27 35,827 7.57 330 2.74 68,809 5.35 326 0.17 34,5218.62 342 2.80 83,281 4.81 328 0.03 30,920 8.48 354 6.23 70,358 4.30 3340.11 30,920 8.17 358 0.19 76,975 4.11 382 2.55 63,920 6.35 387 2.4164,342 6.14 425 2.09 67,198 5.18 436 0.38 66,407 4.76 441 0.22 66,7584.62 442 0.16 66,934 4.53 471 2.81 60,722 6.08 483 1.72 61,204 5.71 4840.27 59,247 5.88 505 2.30 61,526 5.41 506 2.34 60,007 5.42 507 2.7259,928 5.33 510 0.26 54,485 5.27 561 2.52 49,312 6.00 563 3.04 48,0186.20 655 1.85 41,355 6.15 665 0.33 42,243 5.82 719 0.69 39,558 6.55 7592.40 37,116 5.34 825 0.13 35,027 5.10 831 0.53 34,623 4.76 882 0.1030,920 4.94 887 0.24 30,837 4.76 895 0.69 28,893 4.29 908 0.27 25,7536.28 939 0.37 25,851 5.09 941 0.08 22,704 5.15 949 0.21 26,803 4.491,081 2.68 63,836 5.43 1,342 3.74 53,379 4.64The match numbers of the table correspond to the match numbers of the 10% IEF and NEPHGE DBs. The spot numbers given in a previous paper (i 2)to spots aftered in expression by IL-1 B are indicated in parenthesis. *indicates that the spot was not previously found to be aftered inexpression by IL-1 B alone but by other experimental conditions (1 2).For I EF and N EPHG E gels, the analysis was based on 5 DB gels and 3IL-1 B gels and P<0.01 was chosen as level of signfficance. The % IODratio expresses the average % IOD of IL-1 B gels/average % IOD of DBgels. Thus, a ratio above 1 indicates that the spot is upregulated. DNindicates that the spot is synthesized de novo by IL-1 B.

TABLE 11 Optional Proteins Present in Unaffected or Normal Islet CellsIEF 10% gels NEPHGE 10% gels match % IOD match % IOD no. ratio mw pI no.ratio mw pI 10(7−) 11.57 120,478 7.27 9(3) 0.12 115,709 8.33 173(3) 2.47136,657 5.38 18(18) 0.27  36,415 8.44 217(8) 4.73  91,294 6.12 123(8)0.29  57,040 8.17 285(10) 6.01  78,333 6.66 339(12) 0.16  72,602 5.12340(13) 0.32  71,346 5.20 344(11−) 0.44  73,622 4.95 347(14) 4.05 77,651 4.62 480(18) 4.20  62,014 5.91 614(21) 0.27  52,937 4.76 950(26)0.19  25,753 4.53 1,196(2) 9.55 143,064 5.41The match numbers of the table correspond to the match numbers of the10% IEF and NEPHGE DBs. The spot numbers given in a previous paper 20 (i2) to spots aftered in expression by IL-1 B are indicated inparenthesis. * indicates that the spot was not previously found to beaftered in expression by IL-1 B alone but by other experimentalconditions (1 2). For I EF and N EPHG E gels, the analysis was based on5 DB gels and 3 IL-1 B gels and P<0.01 was chosen as level ofsignfficance. The % IOD ratio expresses the average % IOD of IL-1 Bgels/average % IOD of DB gels. Thus, a ratio above I indicates that thespot is upregulated. DN indicates that the spot is synthesized de novoby IL-1 B.

TABLE 12 Optionally Affected Proteins up-/down-regulated or synthesizedde novo by IL-1B Treatment or Expected to be up-/down-regulated orsynthesized de novo in Islet Cells when Immunologically Affected in IDDMIEF 10% gels NEPHGE 10% gels match % IOD match % IOD no. ratio mw pI no.ratio mw pI 10(7−) 11.57 120,478 7.27 9(3) 0.12 115,709 8.33 173(3) 2.47136,657 5.38 18(18) 0.27  36,415 8.44 217(8) 4.73  91,294 6.12 123(8)0.29  57,040 8.17 285(10) 6.01  78,333 6.66 670(1) DN 148,745 8.10339(12) 0.16  72,602 5.12 672(2) DN 132,543 8.52 340(13) 0.32  71,3465.20 673(2) DN 132,204 8.46 344(11−) 0.44  73,622 4.95 674(2) DN 132,5438.42 347(14) 4.05  77,651 4.62 480(18) 4.20  62,014 5.91 614(21) 0.27 52,937 4.76 950(26) 0.19  25,753 4.53 1,196(2) 9.55 143,064 5.41The match numbers of the table correspond to the match numbers of the10% IEF and NEPHGE DBs. The spot numbers given in a previous paper (i 2)to spots aftered in expression by IL-1 B are indicated in parenthesis. *indicates that the spot was not previously found to be aftered inexpression by IL-1 B alone but by other experimental conditions (1 2).For I EF and N EPHG E gels, the analysis was based on 5 DB gels and 3IL-1 B gels and P<0.01 was chosen as level of signfficance. The % IODratio expresses the average % IOD of IL-LB gels/average % IOD of DBgels. Thus, a ratio above I indicates that the spot is upregulated. DNindicates that the spot is synthesized de novo by IL-1 B.

TABLE 13 Optional Marker Proteins in Islet Cells IEF 10% gels NEPHGE 10%gels match % IOD match % IOD no. ratio mw pI no. ratio mw pI 10(7−)11.57 120,478 7.27 9(3) 0.12 115,709 8.33 173(3) 2.47 136,657 5.3818(18) 0.27  36,415 8.44 217(8) 4.73  91,294 6.12 123(8) 0.29  57,0408.17 285(10) 6.01  78,333 6.66 339(12) 0.16  72,602 5.12 340(13) 0.32 71,346 5.20 344(11−) 0.44  73,622 4.95 347(14) 4.05  77,651 4.62480(18) 4.20  62,014 5.91 614(21) 0.27  52,937 4.76 950(26) 0.19  25,7534.53 1,196(2) 9.55 143,064 5.41The match numbers of the table correspond to the match numbers of the 10% IEF and NEPHGE DBs. The spot numbers given in a previous paper (i 2)to spots aftered in expression by IL-1 B are indicated in parenthesis. *indicates that the spot was not previously found to be aftered inexpression by IL-1 B alone but by other experimental conditions (1 2).For I EF and N EPHG E gels, the analysis was based on 5 DB gels and 3IL-1 B gels and P<0.01 was chosen as level of signfficance. The % IODratio expresses the average % IOD of IL-1 B gels/average % IOD of DBgels. Thus, a ratio above I indicates that the spot is upregulated. DNindicates that the spot is synthesized de novo by IL-1 B.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

1. A method for ameliorating the symptoms of diabetes in a subjecthaving diabetes comprising administering to said subject atherapeutically effective amount of a protein comprising humangalectin-3 of SEQ ID NO:4, wherein said protein is a protectivediabetes-mediating protein that ameliorates symptoms of diabetes in saidsubject.
 2. The method according to claim 1, wherein said subject is ahuman.
 3. The method according to claim 1, wherein said diabetes isinsulin-dependent diabetes mellitus.